USOO8932652B2
(12) United States Patent (10) Patent No.: US 8,932,652 B2 Dillon et al. (45) Date of Patent: *Jan. 13, 2015
(54) MICROALGAE-DERIVED COMPOSITIONS (52) U.S. Cl. FOR IMPROVING THE HEALTH AND CPC ...... A23K 1/14 (2013.01); A23L I/1016 APPEARANCE OFSKN (2013.01); A23L 1/3002 (2013.01); A23L I/3004 (2013.01); A23L I/3014 (2013.01); (75) Inventors: Harrison F. Dillon, San Mateo, CA A2.3L I/302 (2013.01); A61K 8/975 (2013.01); (US); Anwar Zaman, South San A61 K3I/715 (2013.01); A61K 36/04 Francisco, CA (US); Anthony G. Day, (2013.01); A61O 19/00 (2013.01); A61O 19/08 San Francsico, CA (US); Anna (2013.01) Coragliotti, San Francisco, CA (US) USPC ...... 424/725 (58) Field of Classification Search (73) Assignee: Solazyme, Inc., South San Francisco, None CA (US) See application file for complete search history. (*) Notice: Subject to any disclaimer, the term of this (56) References Cited patent is extended or adjusted under 35 U.S.C. 154(b) by 0 days. U.S. PATENT DOCUMENTS This patent is Subject to a terminal dis 3,981,996 A 9/1976 Leigh claimer. 3,983,008 A 9, 1976 Shinozaki et al. 4,373,434 A 2, 1983 Alexander et al. 4,417,415 A 1 1/1983 Cysewski et al. (21) Appl. No.: 13/600,102 4,901,635 A 2, 1990 Williams 4,906,746 A 3, 1990 Barnier et al. (22) Filed: Aug. 30, 2012 5,089,481 A 2f1992 Muto et al. 5, 198,217 A 3, 1993 Vedros (65) Prior Publication Data 5,338,673 A 8/1994 Thepenier et al. 5,401,504 A 3, 1995 Das et al. US 2013/OOO4554 A1 Jan. 3, 2013 5,508,033. A 4/1996 Briand 5,521,090 A 5/1996 Donchecket al. 5,643,585 A 7/1997 Arad et al. 5,658,767 A 8/1997 Kyle Related U.S. Application Data 5,680,812 A 10/1997 Linsgeseder (63) Continuation of application No. 1 1/932,782, filed on 5,685,218 A 1 1/1997 Kemper Oct. 31, 2007, which is a continuation of application 5,826,500 A 10/1998 Kemper No. PCT/US2007/001653, filed on Jan. 19, 2007, (Continued) which is a continuation-in-part of application No. 1 1/337,171, filed on Jan. 19, 2006, now abandone FOREIGN PATENT DOCUMENTS and a continuation-in-part of application N EP O996740 B1 9, 2005 1 1/337,103, filed on Jan. 19, 2006, now abandone JP 04-222593. A 8, 1992 and a continuation-in-part of application N 1 1/336,656, filed on Jan. 19, 2006, now abandone (Continued) and a continuation-in-part of application N OTHER PUBLICATIONS 1 1/336,428, filed on Jan. 19, 2006, now abandone and a continuation-in-part of application N “Buffer solution.” Wikipedia, the free encyclopedia, 1-6, (2011), 1 1/336,426, filed on Jan. 19, 2006, now abandone Retrieved from the Intenet May 11, 2011:
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Axenic culutres Technology 38 (1991) 195 are grown in MCY II liquid medium at 2011 See Ramus 1972 25°C. and illuminated with Cool White fluorescent tubes on a 16:8 hr light dark cycle. Cells kept in Suspension by agitation on a gyrorotary shaker or by a stream of filtered air. Porphyridium strain 1380-1a Schmitt D., Water Research unknown See cited reference purpurpet in Volume 35, Issue 3, March 2001, Pages 779-785, Bioprocess Biosyst Eng. 2002 April: 25(1): 35-42. Epub 2002 Mar. 6 Chaetoceros sp. USCE M. A. Guzman-Murillo and unknown See cited reference F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Chiorelia USCE M. A. Guzman-Murillo and unknown See cited reference autotropica F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Chiorelia UTEX58O Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with autotropica Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m2s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Chiorelia UTEXLB2O74 M. A. Guzman-Murillo and Unknown Cultures obtained from various Capsulata F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 (species is through a 0.45 um Millipore filter or a.k.a. Schizochlamydella distilled water depending on capsulata) microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Chiorelia GGMCC S. Guzman, Phytotherapy glucose, Grown in 10 L of membrane filtered Stigmatophora Rscrh (2003) 17: 665-670 glucuronic acid, (0.24 um) seawater and sterilized at xylose, 120° for 30 min and enriched with Erd ribosef fucose Schreiber medium. Cultures maintained at 18 +/- 1. C. under constant 1% CO2 bubbling. Dunaieia DCCBC Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with tertioiecta Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 US 8,932,652 B2 15 16 TABLE 1-continued
Culture and polysaccharide Reported Strain Number purification method Monosaccharide Species Source reference Composition Culture conditions Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Dunaieia DCCBC Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with bardawii Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Isochrysis galbania HCTMS M. A. Guzman-Murillo and unknown Cultures obtained from various var. tahitiana F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Isochrysis galbania UTEXLB 987 Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with war. Tiso Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Isochrysis sp. CCMP7 M. A. Guzman-Murillo and unknown Cultures obtained from various F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Phaeodactylum UTEX 642, 646, M.A.M. A. Guzman-Murillo unknown Cultures obtained from various iricorniitiin 2089 and F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Phaeodactylum GGMCC S. Guzman, Phytotherapy glucose, Grown in 10 L of membrane filtered iricorniitiin Rscrh (2003) 17: 665-670 glucuronic acid, (0.24 um) seawater and sterilized at and mannose 120° for 30 min and enriched with Erd Schreiber medium. Cultures maintained at 18 +/- 1. C. under constant 1% CO2 bubbling. Tetraselmis sp. CCMP 1634 M. A. Guzman-Murillo and unknown Cultures obtained from various 1640; UTEX F. Ascencio., Letters in sources and were cultured in F2 2767 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Botrycoccus UTEX572 and M. A. Guzman-Murillo and unknown Cultures obtained from various brauni 2441 F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Choionococcum UTEX 105 M. A. Guzman-Murillo and unknown Cultures obtained from various F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Hormoilopsis UTEX 104 M. A. Guzman-Murillo and unknown Cultures obtained from various gelatinosa F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or US 8,932,652 B2 17 18 TABLE 1-continued
Culture and polysaccharide Reported Strain Number purification method Monosaccharide Species Source reference Composition Culture conditions distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Neochioris UTEX 1185 M. A. Guzman-Murillo and unknown Cultures obtained from various oleoabundans F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Ochromonas UTEXL1298 M. A. Guzman-Murillo and unknown Cultures obtained from various Danica F. Ascencio., Letters in sources and were cultured in F2 Applied Microbiology 2000, broth prepared with seawater filtered 30, 473–478 through a 0.45 um Millipore filter or distilled water depending on microalgae Salt tolerance. Incubated at 25°C. in flasks and illuminated with white fluorescent lamps. Gyrodinium Yim, Joung Han et. Al., J. of Homopolysaccharide Isolated from seawater collected from inputdictim Microbiol December 2004, 305 of galactose wi red-tide bloom in Korean coastal 14:Yim, J. H. (2000) Ph.D. 2.96% uronic acid water. Maintained in f2 medium at Dissertations, University of 22 under circadian light at Kyung Hee, Seoul 100 uE/m2/sec: dark cycle of 14 h: 10 h. for 19 days. Selected with neomycin and/or cephalosporin 20 ugml Ellipsoidon sp. See cited Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with references Research 44(1999)-67-73; aeration of 100 ml/min and 10% CO2, Lewin, R. A. Cheng, L., for 10s every ten minutes to maintain 1989. Phycologya 28,96 pH > 7.6. Maintained at 22° in 12:12 108 Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Rhodelia UTEX 2320 Talyshinsky, Marina Cancer unknown See Dubinsky O. et al. Composition of reticulata Cell Intl 2002, 2 Cell wall polysaccharide produced by unicellular red algae Rhodella reticulata. 1992 Plant Physiology and biochemistry 30: 409–414 Rhodelia UTEXLB 2506 Evans, L.V., et al. J. Cell Sci Galactose, xylose, Grown in either SWM3 medium or maculata 16, 1-21 (1974); EVANS, L. glucuronic acid ASP12, MgCl2 supplement. 100 mls V. (1970). Br. phycol. J. 5, 1 in 250 mls volumetric Erlenmeyer 13. flask with gentle shaking and 40001X Northern Light fluorescent light for 16 hours. Gymnodinium sp. Oku-1 Sogawa, K., et al., Life unknown See cited reference Sciences, Vol. 66, No. 16, pp. PL 227-231 (2000) AND Umermura, Ken: Biochemical Pharmacology 66 (2003) 481-487 Spiriina platensis UTEXLB 1926 Kaji, Tet. Al.., Life Sci 2002 Na-Sp contains See cited reference Mar. 8: 70(16): 1841-8 two disaccharide Schaeffer and Krylov (2000) repeats: Review-Ectoxicology and Aldobiuronic acid Environmental Safety. 45, and Acofriose + 208-227. other minor Saccharides and Sodium ion Cochiodinium Oku-2 Hasui., et. Al.., Int. J. Bio. mannose, Precultures grown in 500 ml conicals polykrikoides Macromol. Volume 17 No. galactose, glucose containing 300 mls ESM (2) at 21.5°C. 5 1995. and uronic acid for 14 days in continuous light (3500 lux) in growth cabinet) and then transferred to 5 liter conical flask containing 3 liters of ESM. Grown 50 days and then filtered thru wortmann GFF filter. Nostoc miscortin PCCs 7413, Sangar, V K Applied Micro. unknown Growth in nitrogen fixing conditions 7936, 81.13 (1972) & A. M. Burja etal in BG-11 medium in aerated cultures Tetrahydron 57 (2001) 937 maintained in log phase for several 9377; Otero A., J months. 250 mL culture media that Biotechnol. 2003 Apr. were disposed in a temperature 24; 102(2): 143–52 controlled incubator and continuously illuminated with 70 umol photon m-2 S-1 at 30°C. US 8,932,652 B2 19 20 TABLE 1-continued
Culture and polysaccharide Reported Strain Number purification method Monosaccharide Species Source reference Composition Culture conditions Cyanospira See cited A. M. Burja et al. unknown See cited reference capsulata references Tetrahydron 57 (2001) 937 9377 & Garozzo, D., Carbohydrate Res. 1998307 113-124: Ascensio, F. Folia Microbiol (Praha). 2004: 49(1):64–70., Cesaro, A., et al., Int J Biol Macromol. 1990 April; 12(2): 79-84 Cyanothece sp. ATCC S1142 Ascensio F., Folia Microbiol unknown Maintained at 27°C. in ASN III (Praha). 2004: 49(1):64–70. medium with light dark cycle of 16/8 hunder fluorescent light of 3,000 lux light intensity. In Phillips each of 15 strains were grown photoautotrophically in enriched seawater medium. When required the amount of NaNO3 was reduced from 1.5 to 0.35 g/L. Strains axenically grown in an atmosphere of 95% air and 5% CO2 for 8 days under continuous illumination. with mean photon flux of 30 umol photon/m2/s for the first 3 days of growth and 80 umol photonims Chiopeia UTEX 343; Cheng 2004 Journal of unknown See cited reference pyrenoidosa UTEX 1806 Medicinal Food 7(2) 146 152 Phaeodactylum CCAP 1052f1A Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with iricorniitiin Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Chiopeia USCE M. A. Guzman-Murillo and unknown See cited reference autotropica F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Chlorella sp. CCM M. A. Guzman-Murillo and unknown See cited reference F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Dunaieia USCE M. A. Guzman-Murillo and unknown See cited reference tertioiecta F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Isochrysis UTEXLB 987 Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with galabana Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Tetraseinis CCAP 661A-D Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with tetrathele Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, for 10s every ten minutes to maintain pH > 7.6. Maintained at 22° in 12:12 Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) Tetraseinis UTEXLB 2286 M. A. Guzman-Murillo and unknown See cited reference Stiecica F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Tetraseinis CCAP 66.4 Fabregas et al., Antiviral unknown Cultured in 80 ml glass tubes with Stiecica Research 44(1999)-67-73 aeration of 100 ml/min and 10% CO2, and Otero and Fabregas for 10s every ten minutes to maintain Aquaculture 159 (1997) pH > 7.6. Maintained at 22° in 12:12 111-123. Light dark periodicity. Light at 152.3 umol/m2/s. Salinity 3.5% (nutrient enriched as Fabregas, 1984) US 8,932,652 B2 21 22 TABLE 1-continued
Culture and polysaccharide Reported Strain Number purification method Monosaccharide Species Source reference Composition Culture conditions Botrycoccus UTEX 2629 M. A. Guzman-Murillo and unknown See cited reference Sudeticus F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Chlamydomonas UTEX 729 Moore and Tisher Science. unknown See cited reference mexicana 964 Aug. 7: 145:586-7. Dysmorphococci is UTEXLB 65 M. A. Guzman-Murillo and unknown See cited reference globosus F. Ascencio., Letters in Applied Microbiology 2000, 30, 473–478 Rhodelia UTEXLB 2320 S. Geresh et al., J Biochem. unknown See cited reference reticulata Biophys. Methods 50 (2002) 79-187 Review: S. Geresh Biosource Technology 38 (1991) 195-201 Anabena ATCC 29414 Sangar, V K Appl Microbiol. In Vegative wall See cited reference cylindrica 972 November; 24(5): 732-4 where only 18% is carbohydrate-- Glucose 35%), mannose 50%), galactose, xylose, and fucose. In heterocyst wall where 73% is carbohydrate-- Glucose 73% and Mannose is 21% with some galactose and xylose Anabenafios Moore, B G (1965 Can J. Glucose and See cited reference agitae Kingsbury Microbiol. December; 11 (6): 80SE and APPLIED Laboratory, 877-85 ENVIRONMENTAL Cornell MICROBIOLOGY, University April 1978, 718-723) Paineia mucosa See cited Sangar, V K Appl Microbiol. unknown See cited reference references 972 November; 24(5): 732-4; Lewin R.A., (1956) Can J Microbiol. 2: 665-672: Arch Mikrobiol. 1964 Aug. 7:49:158-66 Anacystis PCC 6301 Sangar, V K Appl Microbiol. Glucose, See cited reference nidulians 972 November; 24(5): 732-4 galactose, 80SE Phormidium 94a. See cited Vicente-Garcia V. et al., Galactose, Cultivated in 2 L BG-11 medium at reference Biotechnol Bioeng. 2004 Mannose, 28°C. Acetone was added to Feb. 5: 85(3):306-10 Galacturonic acid, precipitate exopolysaccharide. Arabinose, and Ribose Anabaenaopsis 1402/19 David KA, Fay P. Appl unknown See cited reference circuitaris Environ Microbiol. 1977 December; 34(6): 640-6 Aphanocapsa MN-11 Sudo H., et al., Current Rhamnose; Cultured aerobically for 20 days in halophtia Microobiology Vol. 30 mannose; fucose: seawater-based medium, with 8% (1995), pp. 219-222 galactose; xylose; NaCl, and 40 mg/L NaHPO4. Nitrate glucose In ratio of: change the Exopolysaccharide 15:53:3:3:25 content. Highest cell density was obtaine from culture Supplemented with 100 mg/l NaNO. Phosphorous (40 mg/L) could be added to control the biomass and exopolysaccharide concentration. Aphanocapsa sp See reference De Philippis Ret al., Sci unknown Incubated at 20 and 28°C. with Total Environ. 2005 Nov. 2; artificia light at a photon flux of 5-20 umolm -2s-1. Cylindrotheca sp See reference De Philippis Ret al., Sci Glucuronic acid, Stock enriched cultures incubated at Total Environ. 2005 Nov. 2; Galacturonic acid, 20 and 28°C. with artificial light at a Glucose, photon flux of 5-20 umolm-2s-1. Mannose, Exopolysaccharide production done in Arabinose, glass tu bes containing 100 mL culture Fructose and at 28°C. with continuous illumination Rhamnose at photon density of 5-10 uE m-2s-1. Navicula sp See reference De Philippis Ret al., Sci Glucuronic acid, Incubated at 20 and 28°C. with Total Environ. 2005 Nov. 2; Galacturonic acid, artificia light at a photon flux of 5-20 Glucose, umol m -2s-1. EPS production done in US 8,932,652 B2 23 24 TABLE 1-continued
Culture and polysaccharide Reported Strain Number purification method Monosaccharide Species Source reference Composition Culture conditions Mannose, glass tubes containing 100 mL culture Arabinose, at 28°C. with continuous illumination Fructose and at photon density of 5-10 uE m-2s-1. Rhamnose Gloeocapsa sp See reference De Philippis Ret al., Sci unknown Incubated at 20 and 28°C. with artifical Total Environ. 2005 Nov. 2; light at a photon flux of 5-20 umol m-2s-1. Gloeocapsa See cited Appl Microbiol. unknown See cited references alpicola references 2005: 98(1): 114-20; Photochem Photobiol. 1982 March: 35(3): 359-64; J Gen Microbiol. 1977 anuary: 98(1): 277-80; Arch Microbiol. 1976 February: 107(1): 93-7: FEMS Microbiol Lett. 2002 Sep. 0; 214(2): 229-33 Phaeocystis See cited Toxicology. 2004 Jul. unknown See cited references poucheti references ; 199(2-3): 207-17: Toxicon. 2003 June: 41(7): 803-12; Protist. 2002 September: 153(3): 275-82; J Virol. 2005 July; 79(14): 9236-43; J Bacteriol. 1961 uly: 82(1): 72-9 Leptolyngbya sp See reference De Philippis Ret al., Sci unknown Incubated at 20 and 28°C. with Total Environ. 2005 Nov. 2; artificial light at a photon flux of 5-20 umol m-2s-1. Sympioca sp. See reference De Philippis Ret al., Sci unknown Incubated at 20 and 28°C. with Total Environ. 2005 Nov. 2; artificial light at a photon flux of 5-20 umol m-2s-1. Synechocystis PCC 6714,6803 urgens UJ, Weckesser J. J Glucoseamine, Photoautotrophically grown in BG-11 Bacteriol. 1986 mannosamine, medium, pH 7.5 at 25°C. Mass November; 168(2): 568-73 galactosamine, cultures prepared in a 12 liter mannose and fermentor and gassed by air and glucose carbon dioxide at flow rates of 250 and 2.5 litersh, with illumination from white fluorescent lamps at a constant light intensity of 5,000 lux. Stationeis See reference Lind, J L (1997) Planta unknown See cited reference decipiens 2O3: 213-221 Achnanihes Indiana Holdsworth, R.H., Cell Biol. unknown See cited reference brevipes University 1968 June; 37(3): 831-7: Acta Culture Cient Venez. 2002:53(1): 7 Collection 14.; J. Phycol 36 pp. 882 890 (2000) Achnanihes Strain 330 from Wang, Y., et al., Plant unknown See cited reference longipes National Institute Physiol. 1997 for April; 113(4): 1071-1080. Environmental Studies
Microalgae are preferably cultured in liquid media for 50 bioreactors that contain the fixed carbon source and allow polysaccharide production. Culture condition parameters can light to strike the cells. Such growth is known as heterotrophic be manipulated to optimize total polysaccharide production growth. Any strain of microalgae, including those listed in as well as to alter the structure of polysaccharides produced Table 1, can be cultured in the presence of any one or more by micro algae. fixed carbon source including those listed in Tables 2 and 3. Microalgal culture media usually contains components 55 Such as a fixed nitrogen Source, trace elements, a buffer for pH TABLE 2 maintenance, and phosphate. Other components can include a 2,3-Butanediol fixed carbon Source Such as acetate or glucose, and salts such 2-Aminoethanol as Sodium chloride, particularly for seawater microalgae. 2'-Deoxy Adenosine Examples of trace elements include Zinc, boron, cobalt, cop- 60 3-Methyl Glucose per, manganese, and molybdenum in, for example, the Acetic Acid Adenosine respective forms of ZnCl2, HBO, CoCl2.6H2O. Adenosine-5'-Monophosphate CuCl2.H2O, MnO1.4H2O and (NH), Mo.O.4H2O. Adonitol Some microalgae species can grow by utilizing a fixed Amygdalin carbon source Such as glucose or acetate. Such micro algae 65 Arbutin can be cultured in bioreactors that do not allow light to enter. Bromosuccinic Acid Alternatively, such microalgae can also be cultured in photo US 8,932,652 B2 25 26 TABLE 2-continued TABLE 2-continued Cis-Aconitic Acid L-Ornithine Citric Acid LPhenylalanine D.L-Carnitine L-Proline D.L-Lactic Acid L-Pyroglutamic Acid D.L-C-Glycerol Phosphate L-Rhamnose D-Alanine L-Serine D-Arabitol L-Threonine D-Cellobiose Malonic Acid Dextrin Maltose D-Fructose 10 Maltotriose D-Fructose-6-Phosphate Mannan D-Galactonic Acid Lactone m-Inositol D-Galactose N-Acetyl-DGalactosamine D-Galacturonic Acid N-Acetyl-DGlucosamine D-Gluconic Acid N-Acetyl-LGlutamic Acid D-Glucosaminic Acid 15 N-Acetyl-B-DMannosamine D-Glucose Palatinose D-Glucose-6-Phosphate Phenyethylamine D-Glucuronic Acid p-Hydroxy-Phenylacetic Acid D-Lactic Acid Methyl Ester Propionic Acid D-L-C-Glycerol Phosphate Putrescine D-Malic Acid Pyruvic Acid D-Mannitol Pyruvic Acid Methyl Ester D-Mannose Quinic Acid D-Melezitose Salicin D-Melibiose Sebacic Acid D-Psicose Sedoheptulosan D-Raffinose Stachyose D-Ribose 25 Succinamic Acid D-Saccharic Acid Succinic Acid D-Serine Succinic Acid Mono-Methyl-Ester D-Sorbitol Sucrose D-Tagatose Thymidine D-Trehalose Thymidine-5'-Monophosphate D-Xylose 30 Turanose Formic Acid Tween 40 Gentiobiose Tween 80 Glucuronamide Uridine Glycerol Uridine-5'-Monophosphate Glycogen Urocanic Acid Glycyl-LAspartic Acid Water Glycyl-LGlutamic Acid 35 Xylitol Hydroxy-LProline C-Cyclodextrin i-Erythritol C-D-Glucose nosine C-D-Glucose-1-Phosphate nulin C-D-Lactose taconic Acid C-Hydroxybutyric Acid Lactamide 40 C-Keto Butyric Acid Lactulose C-Keto Glutaric Acid L-Alaninamide C-Keto Valeric Acid L-Alanine C-Ketoglutaric Acid L-Alanylglycine C-Ketovaleric Acid L-Alanyl-Glycine C-Methyl-DGalactoside L-Arabinose 45 C-Methyl-DGlucoside L-Asparagine C-Methyl-DMannoside L-Aspartic Acid B-Cyclodextrin L-Fucose B-Hydroxybutyric Acid L-Glutamic Acid B-Methyl-DGalactoside L-Histidine B-Methyl-D-Glucoside L-Lactic Acid 50 Y-Amino Butyric Acid L-Leucine Y-Hydroxybutyric Acid L-Malic Acid
TABLE 3 (2-amino-3,4-dihydroxy-5-hydroxymethyl-1-cyclohexyl)glucopyranoside (3,4-disinapoyl)fructofuranosyl-(6-Sinapoyl)glucopyranoside (3-Sinapoyl)fructofuranosyl-(6-sinapoyl)glucopyranoside 1 reference 1,10-di-O-(2-acetamido-2-deoxyglucopyranosyl)-2-azi-1,10-decanediol 1,3-mannosylmannose 1,6-anhydrolactose 1,6-anhydrolactose hexaacetate 1,6-dichlorosucrose 1-chlorosucrose 1-desoxy-1-glycinomaltose 1-O-alpha-2-acetamido-2-deoxygalactopyranosyl-inositol 1-O-methyl-di-N-trifluoroacetyl-beta-chitobioside
US 8,932,652 B2 34 TABLE 3-continued O-((Nalpha)-acetylglucosamine 6-sulfate)-(1-3)-idonic acid O-(4-O-feruloyl-alpha-xylopyranosyl)-(1-6)-glucopyranose O-(alpha-idopyranosyluronic acid)-(1-3)-2,5-anhydroalditol-4-sulfate O-(glucuronic acid 2-sulfate)-(1--3)-O-(2,5)-andydrotalitol 6-sulfate O-(glucuronic acid 2-sulfate)-(1-4)-O-(2,5)-anhydromannitol 6-sulfate O-alpha-glucopyranosyluronate-(1-2)-galactose O-beta-galactopyranosyl-(1-4)-O-beta-xylopyranosyl-(1-0)-serine octyl maltopyranoside O-demethylpaulomycin A O-demethylpaulomycin B O-methyl-di-N-acetyl beta-chitobioside Palatinit paldimycin paulomenol A paulomenol B paulomycin A paulomycin A2 paulomycin B paulomycin C paulomycin D paulomycin E paulomycin F phenyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside phenyl O-(2,3,4,6-tetra-O-acetylgalactopyranosyl)-(1-3)-4,6-di-O-acetyl-2-deoxy-2- phthalimido-1-thioglucopyranoside poly-N-4-vinylbenzyllactonamide pseudo-cellobiose pseudo-maltose rhamnopyranosyl-(1-2)-rhamnopyranoside-(1-methyl ether) rhoifolin ruberythric acid S-3105 Senfolomycin A senfolomycin B Solabiose SS SS4 streptobiosamine Sucralfate Sucrose Sucrose acetate isobutyrate Sucrose caproate Sucrose distearate Sucrose monolaurate Sucrose monopalmitate SucroSennonOStearate Sucrose myristate SucroSe Octaacetate Sucrose octabenzoic acid Sucrose octaisobutyrate Sucrose octasulfate Sucrose polyester Sucrose Sulfate Swertiamacroside T-1266 tangshenoside I tetrahydro-2-((tetrahydro-2-furanyl)oxy)-2H-pyran thionigerose Trehalose trehalose 2-sulfate trehalose 6,6'-dipalmitate trehalose-6-phosphate trehallulose trehazolin trichlorosucrose tunicamine turanose
U 77803 xylobiose xylose-glucose xylosucrose
Microalgae contain photosynthetic machinery capable of in the absence of light on a fixed carbon Source that is exog metabolizing photons, and transferring energy harvested enously provided (for example see Plant Physiol. 2005 Feb from photons into fixed chemical energy sources such as ruary; 137(2):460-74). In addition to being a source of chemi monosaccharide. Glucose is a common monosaccharide pro- 65 cal energy, monosaccharides such as glucose are also duced by microalgae by metabolizing light energy and fixing Substrate for production of polysaccharides (see Example carbon from carbon dioxide. Some microalgae can also grow 14). The invention provides methods of producing polysac US 8,932,652 B2 35 36 charides with novel monosaccharide compositions. For turnyellow with occasional shades of light brown, referred to example, microalgae is cultured in the presence of culture as “decolorized biomass'. The invention provides novel media that contains exogenously provided monosaccharide, methods of production of compositions for topical applica Such as glucose. The monosaccharide is taken up by the cell tion including culturing cells of the genus Porphyridium by either active or passive transport and incorporated into under reduced levels of nitrogen (such as, for example, no Tris polysaccharide molecules produced by the cell. This novel and less than 20% of the KNO, per liter of ATCC 1495 ASW method of polysaccharide composition manipulation can be media) and optionally also under relatively light conditions performed with, for example, any microalgae listed in Table 1 such as for example 130 uEms'. In other embodiments, using any monosaccharide or disaccharide listed in Tables 2 the culture media contains no more than 300 mg/L of one or or 3. 10 more nitrate-containing compounds that can be metabolized In some embodiments, the fixed carbon Source is one or by the cells (such as, for example, but not limited to KNO) at more selected from glucose, galactose, Xylose, mannose, inoculation, no more than 250 mg/L at inoculation, no more rhamnose, N-acetylglucosamine, glycerol, floridoside, and than 200 mg/L at inoculation, no more than 175 mg/L at glucuronic acid. The methods may be practiced cell growth in inoculation, no more than 150 mg/L at inoculation, no more the presence of at least about 5.0 LM, at least about 10 uM, at 15 than 135 mg/L at inoculation, no more than 125 mg/L at least about 15.0M, at least about 20.0LM, at least about 25.0 inoculation, no more than 50 mg/L at inoculation, no more uM, at least about 30.0 uM, at least about 35.0 LM, at least than 25 mg/L at inoculation, and no more than 12.5 mg/L at about 40.0 uM, at least about 45.0LM, at least about 50.0LM, inoculation. In some methods the nitrate-containing com at least about 55.0LM, at least about 60.0 uM, at least about pounds are provided in the culture media at inoculation at 75.0 uM, at least about 80.0 uM, at least about 85.0 LM, at approximately 180 mg/L, approximately 160 mg/L, approxi least about 90.0 uM, at least about 95.0 LM, at least about mately 140 mg/L, approximately 130 mg/L, approximately 100.0 LM, or at least about 150.0 LM, of one or more exog 125 mg/L, approximately 110 mg/L, approximately 100 enously provided fixed carbon sources selected from Tables 2 mg/L, and approximately 90 mg/L. In other embodiments, the and 3. culture media contains no more than 300 millimolar of one or In some embodiments using cells of the genus Porphy 25 more nitrate-containing compounds that can be metabolized ridium, the methods include the use of approximately 0.5- by the cells (such as, for example, but not limited to KNO) at 0.75% glycerol as a fixed carbon source when the cells are inoculation, no more than 250 millimolar at inoculation, no cultured in the presence of light. Alternatively, a range of more than 200 millimolar at inoculation, no more than 175 glycerol, from approximately 4.0% to approximately 9.0% millimolar at inoculation, no more than millimolar at inocu may be used when the Porphyridium cells are cultured in the 30 lation, no more than millimolar at inoculation, no more than dark, more preferably from 5.0% to 8.0%, and more prefer millimolar at inoculation, no more than 50 millimolar at ably 7.0%. inoculation, no more than 25 millimolar at inoculation, and After culturing the microalgae in the presence of the exog no more than 12.5 millimolaratinoculation. In some methods enously provided carbon Source, the monosaccharide com the nitrate-containing compounds are provided in the culture position of the polysaccharide can be analyzed as described in 35 media at inoculation at approximately 180 millimolar, Example 5. approximately 160 millimolar, approximately 140 millimo Microalgae culture media can contain a fixed nitrogen lar, approximately 130 millimolar, approximately 125 milli Source Such as KNO. Alternatively, microalgae are placed in molar, approximately 110 millimolar, approximately 100 culture conditions that do not include a fixed nitrogen source. millimolar, and approximately 90 millimolar. Inoculation can For example, Porphyridium sp. cells are cultured for a first 40 mean when seed cells are infused into a bioreactor, and can period of time in the presence of a fixed nitrogen Source, and also mean when cells grown in nitrogen-replete media that then the cells are cultured in the absence of a fixed nitrogen have been pelleted, optionally washed, and are resuspended source (see for example Adda M., Biomass 10:131-140. in culture media that has limiting amounts of nitrogen-con (1986); Sudo H., et al., Current Microbiology Vol.30 (1995), taining compounds or no nitrogen-containing compounds. pp. 219-222; Marinho-Soriano E., Bioresour Technol. 2005 45 Cells inoculated into media containing limiting amounts of February; 96(3):379-82: Bioresour. Technol. 42:141-147 nitrogen-containing compounds, such as 125 mg/L, will typi (1992)). While the invention is not limited by theory, it is well cally undergo cell division until nitrogen is used up (assuming accepted by those skilled in the art that the red color of other nutrients are not limiting), and then begin the “bleach Porphyridium is due to the red pigmented light harvesting ing process in which phycoerythrin is degraded. This pro protein phycoerythrin (for example see Fujimori and Pecci, 50 cess is accelerated as light intensity is increased. Cells grown Distinct subunits of phycoerythrin from Porphyridium cruen in nitrogen-replete media can also be harvested and washed tum and their spectral characteristics, Arch. Biochem. Bio and resuspended in culture media that contains no nitrogen or phys. 118, 448-55 1967). Culture of Porphyridium in the a limited amount of nitrogen such as the amounts listed above. presence of reduced levels of nitrogen causes cells to degrade In addition, nitrogen-replete media can be exchanged with phycoerythrin, resulting in a significant decrease in the 55 nitrogen-limited media through tangential flow filtration amount of red pigmentation. Because phycoerythrin consti using a filter that has a pore size Smaller than the diameter of tutes over 2% of the dry weight of Porphyridium cells under the cells. The diameter of Porphyridium cells is approxi nitrogen-replete conditions (see for example M. M. Rebol mately 4-8 microns. This method avoids centrifugation and loso Fuentes 2000, Food Chemistry, 70; 345-353), this cata reduces the possibility of contamination. bolic process allows a significant amount of fixed nitrogen to 60 Other methods include removing red coloration from dif be recycled. Again while the invention is not limited by ferent species of microalgae through mutagenesis. For theory, providing excess light also causes Porphyridium cells example, species of the genus Porphyridium are subjected to to degrade phycoerythrin to reduce the amount of light har chemical mutagenesis, followed by screening for colonies vesting per cell. This process reduces oxidative stress caused lacking red coloration. See for example Sivan and Arad, Phy by excess photon flux in the thylakoid membrane. Porphy 65 cologia 32(1), pp. 68-72 (1993). Such genetically decolorized ridium biomass grown in nitrogen-limited conditions, par strains are used to generate non-red biomass for formulation ticularly when grown under highlight, lose redcoloration and as skin care products. In a preferred embodiment the geneti US 8,932,652 B2 37 38 cally decolorized biomass is homogenized. In another pre ond, it is also known that sulfate can inhibit the uptake of ferred embodiment the polysaccharide contains more than metal ions required for nitrogen fixation such as molybde 4.75% sulfur by weight. While the invention is not limited by num, and that increasing Sulfate concentrations negatively theory, production of phycoerythrin is reduced in some affects algae Such as cyanobacteria even at Sulfate concentra mutagenized strains due to mutations in various regions of the tions in estuarine (>8-10 mM) and seawater (28 mM) levels of genome including promoters, coding regions, and other func sufate (see for example Marino et al. Hydrobiologia 500: pp. tional elements. Both bleaching through nutrient limitation 277-293, 2004). Third, sulfate at high levels can often be and excess light, as well as through mutagenesis, can be taken up and reduced and Sulfide, which is toxic to photosyn performed on any microalgae species, including those listed thesis because it attacks photosystem II (see for example in Table 1. 10 Khanal et al., J. Envir. Engrg., 129(12); pp. 1104-11 11, 2003). Some methods further comprise formulating decolorized Fourth, high sulfate levels alter the osmotic pressure of the Porphyridium biomass, generated through any or all of the growth media, and many organisms cannot survive at Such an methods nutrient limitation, excess light, and mutagenesis, elevated osmoticum. For example, it is well established that with a carrier suitable for topical administration. The methods photosynthesis of algae is inhibited by hyperosmotic and salt also optionally include formulating decolorized Porphy 15 stresses. See for example “Suppression of Quantum Yield of ridium biomass with one or more preservatives, such as for Photosystem II by Hyperosmotic Stress in Chlamydomonas example diiodomethyl-p-tolylsulfone, 2-Bromo-2-nitropro reinhardtii Plant Cell Physiol. 36: pp. 1253-1258 (1995); pane-1,3-diol, cis isomer 1-(3-chloroallyl)-3,5,7-triaza-1- and “The Effect of Osmotic and Ionic Stress on the Primary azoniaadamantane chloride (a.k.a. Dowicil 200), glutaralde Processes of Photosynthesis in Dunaliella tertiolecta', J. hyde, 4,4-dimethyl oxazolidine, 7-Ethylbicyclooxazolidine, Exp. Bot. 1984 35(1): 18-27 (1984). methyl paraben, Sorbic acid, methyl paraben, Germaben II, By use of methods described above, the disclosed inven and disodium EDTA. tion includes a preparation of cells, cell biomass, or cell Other culture parameters can also be manipulated, such as homogenate containing a reduced level of green pigmenta the pH of the culture media, the identity and concentration of tion, or a reduced absorbance at 545 nm, relative to the same trace elements such as those listed in Table 3, and other media 25 cells grown under different conditions. The cells may be those constituents. One non-limiting example is the inclusion of at of any microalgae as described herein, including those of the least one source of sulfate in the culture media to increase the genus Porphyridium. The cells, cell biomass, or cell homo level of Sulfation in the polysaccharides produced. In some genate may be formulated into a composition of the disclo embodiments, a polysaccharide preparation method is prac Sure Such that an aqueous extract of the composition would ticed with culture media containing more than about 100, 30 contain the same reduced level of green pigmentation. more than about 150, more than about 200, more than about In some embodiments, an aqueous extract of the composi 250, more than about 300, more than about 350, more than tion contains no more than about 75%, no more than about about 400, more than about 450, more than about 500, more 70%, no more than about 65%, no more than about 60%, no than about 550, more than about 600, more than about 650, more than about 55%, no more than about 50%, no more than more than about 700, or more thanabout 750, more thanabout 35 about 45%, no more than about 40%, no more than about 800, more than about 850, more than about 900, more than 35%, no more than about 30%, no more than about 25%, no about 950, or more than about 1 or 2 M sulfate (or total more than about 20%, no more than about 15%, no more than SO). Increasing the sulfation has been demonstrated to about 10%, or no more than about 5% of the absorbance per increase the antioxidant apacity of the polysaccharide (see gram at 545 nm compared to an extract of cells of the same example 23 and Spitz et al. (J. Appl. Phycology (2005) 40 species grown in a photobioreactor in ATCC 1495 ASW 17:215-222). Without being bound by theory, and offered to media (as described in Example 1) in the presence of 50 improved the understanding of certain aspects of the dis microeinsteins of light per second per square meter. One closed invention, it is possible that an increased level of non-limiting means for detection of absorbance is by use of a Sulfation may increase the anti-cholesterol characteristics of spectrophotometer. the homogenized cell material or polysaccharide preparation 45 Microalgae can be grown in the presence of light. The disclosed herein. The correlation between higher amounts of number of photons striking a culture of microalgae cells can Sulfation and antioxidant activity demonstrated herein was be manipulated, as well as other parameters such as the wave unexpected given the weak antioxidant activity of carrag length spectrum and ratio of dark:light hours per day. eenan, which contains as much as 40% sulfate. Microalgae can also be cultured in natural light, as well as It is believed that microalgae of the genus Porphyridium 50 simultaneous and/or alternating combinations of natural light have not been grown or propagated under conditions with and artificial light. For example, microalgae of the genus sulfate concentrations of 100 mM to 2 M. Thus the invention Chlorella be cultured under natural light during daylight includes the Surprising discovery that microalgae are capable hours and under artificial light during night hours. of growth under Such conditions. Additionally, the invention The gas content of a photobioreactor can be manipulated. is based in part on the Surprising discovery that growth under 55 Part of the volume of a photobioreactor can contain gas rather higher Sulfate concentrations can produce polysaccharides than liquid. Gas inlets can be used to pump gases into the with higher levels of sulfation. This allows for the production photobioreactor. Any gas can be pumped into a photobiore of cells (and so biomass) containing highly sulfated polysac actor, including air, air/CO mixtures, noble gases such as charides that may be used in the form of purified polysaccha argon and others. The rate of entry of gas into a photobiore rides, a homogenate of cells (biomass), intact cells (biomass) 60 actor can also be manipulated. Increasing gas flow into a per se, or a combination thereof. photobioreactor increases the turbidity of a culture of The discovery that Porphyridium can survive above 100 microalgae. Placement of ports conveying gases into a pho mM Sulfate was Surprising for a number of reasons. First, it is tobioreactor can also affect the turbidity of a culture at a given known that sulfate can alter the rate of uptake of toxic metal gas flow rate. Air/CO mixtures can be modulated to generate ions in algae, Such as chromium, and that increasing metal 65 different polysaccharide compositions by manipulating car accumulation can lead to toxicity (see for example Kaszycki bon flux. For example, air:CO mixtures of about 99.75% et al, Plant, Cell & Environment, 28(2): p. 260, 2005). Sec air:0.25% CO, about 99.5% air:0.5% CO, about 99.0% US 8,932,652 B2 39 40 air:1.00% CO, about 98.0% air:2.0% CO, about 97.0% increase in number; however quantities of aqueous culture air:3.0% CO, about 96.0% air:4.0% CO, and about 95.00% medium are not flowed through the photobioreactor through air:5.0% CO can be infused into a bioreactor or photobiore out the time period. Thus in some embodiments, aqueous actOr. culture medium is not flowed through the photobioreactor Microalgae cultures can also be subjected to mixing using after inoculation. devices such as spinning blades and propellers, rocking of a In other instances culture media can be flowed though the culture, stir bars, and other instruments. photobioreactor throughout the time period during which the B. Cell Culture Methods: Photobioreactors microalgae reproduce and increase in number. In some Microalgae can be grown and maintained in closed photo instances media is infused into the photobioreactor after bioreactors made of different types of transparent or semi 10 inoculation but before the cells reach a desired density. In transparent material. Such material can include Plexiglas(R) other words, a turbulent flow regime of gas entry and media enclosures, glass enclosures, bags bade from Substances Such entry is not maintained for reproduction of microalgae until a as polyethylene, transparent or semitransparent pipes, and desired increase in number of said microalgae has been other materials. Microalgae can also be grown in open ponds. achieved, but instead a parameter Such as gas entry or media Photobioreactors can have ports allowing entry of gases, 15 entry is altered before the cells reach a desired density. Solids, semisolids and liquids into the chamber containing the Photobioreactors preferably have one or more ports that microalgae. Ports are usually attached to tubing or other allow gas entry. Gas can serve to both provide nutrients such means of conveying Substances. Gas ports, for example, con as CO as well as to provide turbulence in the culture media. vey gases into the culture. Pumping gases into a photobiore Turbulence can be achieved by placing a gas entry port below actor can serve to both feed cells CO and other gases and to the level of the aqueous culture media so that gas entering the aerate the culture and therefore generate turbidity. The photobioreactor bubbles to the surface of the culture. One or amount of turbidity of a culture varies as the number and more gas exit ports allow gas to escape, thereby preventing position of gas ports is altered. For example, gas ports can be pressure buildup in the photobioreactor. Preferably a gas exit placed along the bottom of a cylindrical polyethylene bag. port leads to a “one-way valve that prevents contaminating Microalgae grow faster when CO is added to air and bubbled 25 microorganisms to enter the photobioreactor. In some into a photobioreactor. For example, a 5% CO:95% air mix instances cells are cultured in a photobioreactor for a period ture is infused into a photobioreactor containing cells of the of time during which the microalgae reproduce and increase genus Porphyridium (see for example Biotechnol Bioeng. in number, however a turbulent flow regime with turbulent 1998 Sep. 20; 59(6):705-13; textbook from office: U.S. Pat. eddies predominantly throughout the culture media caused by Nos. 5,643,585 and 5,534,417; Lebeau, T., et. al. Appl. 30 gas entry is not maintained for all of the period of time. In Microbiol. Biotechnol (2003) 60:612-623; Muller-Fuega, A., other instances a turbulent flow regime with turbulent eddies Journal of Biotechnology 103 (2003 153-163; Muller-Fuega, predominantly throughout the culture media caused by gas A., Biotechnology and Bioengineering, Vol. 84, No. 5, Dec. 5, entry can be maintained for all of the period of time during 2003; Garcia-Sanchez, J. L., Biotechnology and Bioengi which the microalgae reproduce and increase in number. In neering, Vol. 84, No. 5, Dec. 5, 2003; Garcia-Gonzales, M., 35 Some instances a predetermined range of ratios between the Journal of Biotechnology, 115 (2005) 81-90. Molina Grima, scale of the photobioreactor and the scale of eddies is not E., Biotechnology Advances 20 (2003) 491-515). maintained for the period of time during which the microal Photobioreactors can be exposed to one or more light gae reproduce and increase in number. In other instances Such Sources to provide microalgae with light as an energy source a range can be maintained. via light directed to a surface of the photobioreactor. Prefer 40 Photobioreactors preferably have at least one port that can ably the light source provides an intensity that is sufficient for be used for sampling the culture. Preferably a sampling port the cells to grow, but not so intense as to cause oxidative can be used repeatedly without altering compromising the damage or cause a photoinhibitive response. In some axenic nature of the culture. A sampling port can be config instances a light Source has a wavelength range that mimics or ured with a valve or other device that allows the flow of approximately mimics the range of the Sun. In other instances 45 sample to be stopped and started. Alternatively a sampling a different wavelength range is used. Photobioreactors can be port can allow continuous sampling. Photobioreactors pref placed outdoors or in a greenhouse or other facility that erably have at least one port that allows inoculation of a allows sunlight to strike the surface. Photon intensities are culture. Such a port can also be used for other purposes Such typically measured in microeinsteins of light per square meter as media or gas entry. per second (uEms) although other measurements such as 50 Microalgae that produce polysaccharides can be cultured lux and footcandles are sometimes used. Preferred photon in photobioreactors. Microalgae that produce polysaccharide intensities for culturing species of the genus Porphyridium that is not attached to cells can be cultured for a period of time are between 50 and 300 uEm’s' (see for example Photo and then separated from the culture media and secreted synth Res. 2005 June; 84(1-3):21-7), although in cases of polysaccharide by methods such as centrifugation and tan inducing Porphyridium cells to degrade phycoerythrin pre 55 gential flow filtration. Preferred organisms for culturing in ferred light intensities can be higher, such as for example photobioreactors to produce polysaccharides include Porphy 400-700 uEm’s'. ridium sp., Porphyridium Cruentum, Porphyridium pur Photobioreactors preferably have one or more parts that pureum, Porphyridium aerugineum, Rhodella maculata, allow media entry. It is not necessary that only one Substance Rhodella reticulata, Chlorella autotrophica, Chlorella Stig enter or leave a port. For example, a port can be used to flow 60 matophora, Chlorella capsulata, Achnanthes brevipes, Ach culture media into the photobioreactor and then later can be nanthes longzpes, Gloeocapsa alpicola and Phaeocysstis used for sampling, gas entry, gas exit, or other purposes. In pouchettii. some instances a photobioreactor is filled with culture media C. Non-Microalgal Polysaccharide Production at the beginning of a culture and no more growth media is Organisms besides microalgae can be used to produce infused after the culture is inoculated. In other words, the 65 polysaccharides, such as lactic acid bacteria (see for example microalgal biomass is cultured in an aqueous medium for a Stinglee, F., Molecular Microbiology (1999) 32(6), 1287 period of time during which the microalgae reproduce and 1295: Ruas Madiedo, P. J. Dairy Sci. 88:843-856 (2005): US 8,932,652 B2 41 42 Laws, A., Biotechnology Advances 19 (2001) 597–625; Xan at least about 0.12 micrometer, at least about 0.14 microme than gum bacteria: Pollock, T.J., J. Ind. Microbiol Biotech ter, at least about 0.16 micrometer, at least about 0.18 nol., 1997 August; 19(2): 92-7. Becker, A., Appl. Micrbiol. micrometer, at least about 0.2 micrometer, at least about 0.22 Bioltechnol. 1998 August: 50(2): 92-7: Garcia-Ochoa, F., micrometer, or at least about 0.45 micrometer. Preferred pore Biotechnology Advances 18 (2000) 549-579., seaweed: sizes of TFF allow contaminants to pass through but not Talarico, L. B., et al., Antiviral Research 66 (2005) 103-110: polysaccharide molecules. Dussealt, J., et al., J Biomed Mater Res A. (2005) Nov. 1; Ion Exchange Chromatography Melo, F. R., J Biol Chem 279:20824-35 (2004)). Anionic polysaccharides can be purified by anion D. Ex Vivo Methods exchange chromatography. (Jacobsson, I., Biochem J. 1979 Microalgae and other organisms can be manipulated to 10 produce polysaccharide molecules that are not naturally pro Apr. 1; 179(1):77-89: Karamanos, N. K., Eur J. Biochem. duced by methods such as feeding cells with monosaccha 1992 Mar. 1; 204(2):553–60). rides that are not produced by the cells (Nature. 2004 Aug. 19: Protease Treatment 430(7002):873-7). For example, species listed in Table 1 are Polysaccharides can be treated with proteases to degrade grown according to the referenced growth protocols, with the 15 contaminating proteins. In some instances the contaminating additional step of adding to the culture media a fixed carbon proteins are attached, either covalently or noncovalently to source that is not in the culture media as published and ref polysaccharides. In other instances the polysaccharide mol erenced in Table 1 and is not produced by the cells in a ecules are in a preparation that also contains proteins. Pro detectable amount. teases can be added to polysaccharide preparations contain E. In vitro Methods ing proteins to degrade proteins (for example, the protease Polysaccharides can be altered by enzymatic and chemical from Streptomyces griseus can be used (Sigma Aldrich cata modification. For example, carbohydrate modifying enzymes log number P5147). After digestion, the polysaccharide is can be added to a preparation of polysaccharide and allowed preferably purified from residual proteins, peptide fragments, to catalyze reactions that alter the structure of the polysac and amino acids. This purification can be accomplished, for charide. Chemical methods can be used to, for example, 25 example, by methods listed above Such as dialysis, filtration, modify the sulfation pattern of a polysaccharide (see for and precipitation. example Carbohydr. Polym. 63:75-80 (2000); Pomin V H., Heat treatment can also be used to eliminate proteins in Glycobiology. 2005 December; 15(12): 1376-85; Naggi A. polysaccharide preparations (see for example Biotechnol Semin Thromb Hemost. 2001 October; 27(5):437-43 Lett. 2005 January; 27(1): 13-8: FEMS Immunol Med. Micro Review: Habuchi, O., Glycobiology. 1996 January; 6(1): 30 biol. 2004 Oct. 1; 42(2):155-66; Carbohydr Res. 2000 Sep. 8: 51-7: Chen, J. J. Biol. Chem. In press; Geresh. Set al., J. 328(2):199-207; J Biomed Mater Res. 1999; 48(2):111-6.: Biochem. Biophys. Methods 50 (2002) 179-187). Carbohydr Res. 1990 Oct. 15: 207(1): 101-20;). F. Polysaccharide Purification Methods The invention thus includes production of an exopolysac Exopolysaccharides can be purified from microalgal cul charide comprising separating the exopolysaccharide from tures by various methods, including those disclosed herein. 35 Precipitation contaminants after proteins attached to the exopolysaccha For example, polysaccharides can be precipitated by add ride have been degraded or destroyed. The proteins may be ing compounds such as cetylpyridinium chloride, isopro those attached to the exopolysaccharide during culture of a panol, ethanol, or methanol to an aqueous solution containing microalgal cell in media, which is first separated from the a polysaccharide in Solution. Pellets of precipitated polysac 40 cells prior to proteolysis or protease treatment. The cells may charide can be washed and resuspended in water, buffers such be those of the genus Porphyridium as a non-limiting as phosphate buffered saline or Tris, or other aqueous solu example. tions (see for example Farias, W. R. L., et al., J. Biol. Chem. In one non-limiting example, a method of producing an (2000) 275; (38)29299-29307; U.S. Pat. Nos. 6,342,367; exopolysaccharide is provided wherein the method com 6,969,705). 45 prises culturing cells of the genus Porphyridium; separating Dialysis cells from culture media; destroying protein attached to the Polysaccharides can also be dialyzed to remove excess salt exopolysaccharide present in the culture media; and separat and other Small molecules (see for example Gloaguen, V., et ing the exopolysaccharide from contaminants. In some meth al., Carbohydr Res. 2004 Jan. 2: 339(1): 97-103; Microbiol ods, the contaminant(s) are selected from amino acids, pep Immunol. 2000; 44(5):395-400.). 50 tides, proteases, protein fragments, and salts. In other Tangential Flow Filtration methods, the contaminant is selected from NaCl, MgSO, Filtration can be used to concentrate polysaccharide and MgCl, CaCl, KNO. KHPO, NaHCO, Tris, ZnCl2. remove salts. For example, tangential flow filtration (TFF), HBO, CoCl2, CuCl2, MnCl2 (NH), Mo.O. FeCl3 and also known as cross-flow filtration, can be used)). For a pre EDTA ferred filtration method see Geresh, Carb. Polym. 50: 183 55 Drying Methods 189 (2002), which discusses use of a MaxCell A/G technolo After purification of methods such as those above, polysac gies 0.45 uM hollow fiber filter. Also see for example charides can be dried using methods such as lyophilization Millipore Pellicon(R) devices, used with 100kD,300 kD, 1000 and heat drying (see for example Shastry, S., Brazilian Jour kD (catalog number P2C01MC01), 0.1 uM (catalog number nal of Microbiology (2005) 36:57-62: Matthews KH., Int J. P2VVPPV01), 0.22 uM (catalog number P2GVPPV01), and 60 Pharm. 2005 Jan. 31: 289(1-2):51-62. Epub 2004 Dec. 30: 0.45 uM membranes (catalog number P2HVMPV01). It is Gloaguen, V., et al., Carbohydr Res. 2004 Jan. 2: 339(1): 97 preferred that the polysaccharides do not pass through the 103). filter at a significant level. It is also preferred that polysac Tray dryers accept moist Solid on trays. Hot air (or nitro charides do not adhere to the filter material. TFF can also be gen) can be circulated to dry. Shelf dryers can also employ performed using hollow fiber filtration systems. 65 reduced (below atmospheric at sea level, such as at about 25 Non-limiting examples of tangential flow filtration include in Hg or less) pressure or vacuum to dry at room temperature use of a filter with a pore size of at least about 0.1 micrometer, when products are temperature sensitive and are similar to a US 8,932,652 B2 43 44 freeze-drier but less costly to use and can be easily scaled-up. Homogenization as described herein can increase the In some embodiments drying in oven tray dryers is performed amount of solvent-available polysaccharide significantly. For under vacuum. example, homogenization can increase the amount of Sol Spray dryers are relatively simple in operation, which vent-available polysaccharide by at least a factor of 0.25, at accept feed in fluid state and convert it into a dried particulate least a factor of 0.5, at least a factor of 1, at least a factor of 2, form by spraying the fluid into a hot drying medium. at least a factor of 3, at least a factor of 4, at least a factor of 5, Rotary dryers operate by continuously feeding wet mate at least a factor of 8, at least a factor of 10, at least a factor of rial, which is dried by contact with heated air, while being 15, at least a factor of 20, at least a factor of 25, and at least a transported along the interior of a rotating cylinder, with the factor of 30 or more compared to the amount of solvent rotating shell acting as the conveying device and stirrer. 10 available polysaccharide in an identical or similar quantity of Spin flash dryers are used for drying of wet cake, slurry, or non-homogenized cells of the same type. One way of deter paste which is normally difficult to dry in other dryers. The mining a quantity of cells Sufficient to generate a given quan material is fed by a screw feeder through a variable speed tity of homogenate is to measure the amount of a compound drive into the vertical drying chamber where it is heated by air in the homogenate and calculate the gram quantity of cells and at the same time disintegrated by a specially designed 15 required to generate this amount of the compound using disintegrator. The heating of air may be direct or indirect known data for the amount of the compound per gram mass of depending upon the application. The dry powder is collected cells. The quantity of many such compounds per gram of through a cyclone separator/bag filter or with a combination particular microalgae cells are know. For examples, see FIG. of both. 7. Given a certain quantity of a compound in a composition, Whole Cell Extraction the skilled artisan can determine the number of grams of Intracellular polysaccharides and cell wall polysaccha intact cells necessary to generate the observed amount of the rides can be purified from whole cell mass (see form example compound. The number of grams of microalgae cells present U.S. Pat. No. 4,992,540; U.S. Pat. No. 4,810,646; J Sietsma J in the composition can then be used to determine if the com H., et al., Gen Microbiol. 1981 July; 125(1):209-12: Fleet G position contains at least a certain amount of Solvent-avail H. Manners D.J., J Gen Microbiol. 1976 May: 94(1): 180-92). 25 able polysaccharide sufficient to indicate whether or not the G. Microalgae Homogenization Methods composition contains homogenized cells. Such as for example A pressure disrupter pumps of a slurry through a restricted five times the amount of solvent-available polysaccharide orifice valve. High pressure (up to 1500 bar) is applied, fol present in a similar or identical quantity of unhomogenized lowed by an instant expansion through an exiting nozzle. Cell cells. disruption is accomplished by three different mechanisms: 30 H. Analysis Methods impingement on the valve, high liquid shearin the orifice, and Assays for detecting polysaccharides can be used to quan Sudden pressure drop upon discharge, causing an explosion of titate starting polysaccharide concentration, measure yield the cell. The method is applied mainly for the release of during purification, calculate density of secreted polysaccha intracellular molecules. According to Hetherington et al., cell ride in a photobioreactor, measure polysaccharide concentra disruption (and consequently the rate of protein release) is a 35 tion in a finished product, and other purposes. first-order process, described by the relation: log Rm/(Rm The phenol: sulfuric acid assay detects carbohydrates (see R)=KN P72.9. R is the amount of soluble protein; Rim is the Hellebust, Handbook of Phycological Methods, Cambridge maximum amount of soluble protein K is the temperature University Press, 1978; and Cuesta G. et al., J. Microbiol dependent rate constant: N is the number of passes through Methods. 2003 January: 52(1):69-73). The 1.6 dimethylm the homogenizer (which represents the residence time). P is 40 ethylene blue assay detects anionic polysaccharides. (see for the operating pressure. example BrazJ Med Biol Res. 1999 May; 32(5):545-50; Clin In a ball mill, cells are agitated in Suspension with Small Chem. 1986 November; 32(11):2073-6). abrasive particles. Cells break because of shear forces, grind Polysaccharides can also be analyzed through methods ing between beads, and collisions with beads. The beads Such as HPLC, size exclusion chromatography, and anion disrupt the cells to release biomolecules. The kinetics of 45 exchange chromatography (see for example Prosky L, Asp N. biomolecule release by this method is also a first-order pro Schweizer T F. DeVries J W & Furda I (1988) Determination CCSS, of insoluble, soluble and total dietary fiber in food and food Another widely applied method is the cell lysis with high products: Interlaboratory study. Journal of the Association of frequency Sound that is produced electronically and trans Official Analytical Chemists 71, 1017+1023; IntJ Biol Mac ported through a metallic tip to an appropriately concentrated 50 romol. 2003 November; 33(1-3):9-18) cellular Suspension, i.e.: Sonication. The concept of ultrasonic Polysaccharides can also be detected using gel electro disruption is based on the creation of cavities in cell Suspen phoresis (see for example Anal Biochem. 2003 Oct. 15: 321 sion. Homogenization can also be performed with a Microf (2):174-82; Anal Biochem. 2002 Jan. 1; 300(1):53-68). luidizerR) device (such as the M-1 10Y Microfluidizer(R) Monosaccharide analysis of polysaccharides can be per model, Microfluidics Inc., Newton, Mass.). 55 formed by combined gas chromatography/mass spectrometry Blending (high speed or Waring), the french press, or even (GC/MS) of the per-O-trimethylsilyl (TMS) derivatives of the centrifugation in the case of weak cell walls, also disrupt the monosaccharide methyl glycosides produced from the cells by using the same concepts. sample by acidic methanolysis (see Merkle and Poppe (1994) Cells can also be ground after drying in devices such as a Methods Enzymol. 230:1-15; York, et al. (1985) Methods colloid mill. 60 Enzymol. 118:3-40). Because the percentage of polysaccharide as a function of The determination of protein concentration may be by use the dry weight of a microalgae cell can frequently be in excess of any known procedure, such as the Lowry assay, the Biuret of 50%, microalgae cell homogenates can be considered par assay, the Bradford assay, or the bicinchoninic acid (BCA) tially pure polysaccharide compositions. Cell disruption aids assay. As a non-limiting example, the BCA assay is based on in increasing the amount of solvent-accessible polysaccha 65 the formation of a Cu"-protein complex under alkaline con ride by breaking apart cell walls that are largely composed of ditions. The Cu" is then reduced to Cu'" where the amount of polysaccharide. protein present is proportional to the amount reduction. The US 8,932,652 B2 45 46 reduction has been shown to be mediated by amino acids Such amount of a monosaccharide may also be expressed in terms as cysteine, cystine, tryptophan, and tyrosine as well as the of an increase relative to other monosaccharides rather than peptide bond. The result of the assay is a purple-blue complex relative to the unit mass, or mole, of polysaccharide. An with Cu" under alkaline conditions. The color complex is example of genetic modification leading to production of stable, even in the presence of other components possibly 5 modified polysaccharides is transforming a microalgae with a present with the proteins, such as detergents. The amount of carbohydrate transporter gene, and culturing a transformant reduction can be monitored by absorbance at 562 nm. The in the presence of a monosaccharide which is transported into BCA assay is sensitive and accurate over a broad range of the cell from the culture media by the carbohydrate trans protein concentrations. porter protein encoded by the carbohydrate transporter gene. III Compositions 10 In some instances the culture can be in the dark, where the A. General monosaccharide, Such as glucose, is used as the Sole energy Compositions of the invention include a microalgal source for the cell. In other instances the culture is in the light, polysaccharide or homogenate as described herein. In where the cells undergo photosynthesis and therefore pro embodiments relating to polysaccharides, including duce monosaccharides such as glucose in the chloroplast and exopolysaccharides, the composition may comprise a 15 transport the monosaccharides into the cytoplasm, while homogenous or a heterogeneous population of polysaccha additional exogenously provided monosaccharides are trans ride molecules, including Sulfated polysaccharides as non ported into the cell by the carbohydrate transporter protein. In limiting embodiments. Non-limiting examples of homog both instances monosaccharides from the cytoplasm are enous populations include those containing a single type of transported into the endoplasmic reticulum, where polysac polysaccharide molecule, Such as that with the same structure charide synthesis occurs. Novel polysaccharides produced by and molecular weight. Non-limiting examples of heteroge non-genetically engineered microalgae can therefore be gen neous populations include those containing more than one erated by nutritional manipulation, i.e.: exogenously provid type of polysaccharide molecule. Such as a mixture of ing carbohydrates in the culture media that are taken up polysaccharides having a molecular weight (MW) within a through endogenous transport mechanisms. Uptake of the range or a MW above or below a MW value. For example, the 25 exogenously provided carbohydrates can be induced, for Porphyridium sp. exopolysaccharide is typically produced in example, by culturing the cells in the dark, thereby forcing the a range of sizes from 3-5 million Daltons. Of course a cells to utilize the exogenously provided carbon source. For polysaccharide containing composition of the invention may example, Porphyridium cells cultured in the presence of 7% be optionally protease treated, or reduced in the amount of glycerol in the dark produce a novel polysaccharide because protein, as described above. 30 the intracellular carbon flux under these nutritionally manipu In Some embodiments, a composition of the invention may lated conditions is different from that under photosynthetic comprise one or more polysaccharides produced by microal conditions. Insertion of carbohydrate transporter genes into gae that have not been recombinantly modified. The microal microalgae facilitates, but is not strictly necessary for, gae may be those which are naturally occurring or those polysaccharide structure manipulation because expression of which have been maintained in culture in the absence of 35 Such genes can significantly increase the concentration of a alteration by recombinant DNA techniques or genetic engi particular monosaccharide in the cytoplasm of the cell. Many neering. carbohydrate transporter genes encode proteins that transport In other embodiments, the polysaccharides are those from more than one monosaccharide, albeit with different affinities modified microalgae, such as, but not limited to, microalgae for different monosaccharides (see for example Biochimica et modified by recombinant techniques. Non-limiting examples 40 Biophysica Acta 1465 (2000) 263-274). In some instances a of Such techniques include introduction and/or expression of microalgae species can be transformed with a carbohydrate an exogenous nucleic acid sequence encoding a gene product: transporter gene and placed under different nutritional con genetic manipulation to decrease or inhibit expression of an ditions, wherein one set of conditions includes the presence of endogenous microalgal gene product; and/or genetic manipu exogenously provided galactose, and the other set of condi lation to increase expression of an endogenous microalgal 45 tions includes the presence of exogenously provided Xylose, gene product. The invention contemplates recombinant and the transgenic species produces structurally distinct modification of the various microalgae species described polysaccharides under the two conditions. By altering the herein. In some embodiments, the microalgae is from the identity and concentration of monosaccharides in the cyto genus Porphyridium. plasm of the microalgae, through genetic and/or nutritional Polysaccharides provided by the invention that are pro 50 manipulation, the invention provides novel polysaccharides. duced by genetically modified microalgae or microalgae that Nutritional manipulation can also be performed, for example, are provided with an exogenous carbon Source can be distinct by culturing the microalgae in the presence of high amounts from those produced by microalgae cultured in minimal of Sulfate, as described herein. In some instances nutritional growth media under photoautotrophic conditions (i.e.: in the manipulation includes addition of one or more exogenously absence of a fixed carbon Source) at least in that they contain 55 provided carbon Sources as well as one or more other non a different monosaccharide content relative to polysaccha carbohydrate culture component, such as 50 mM MgSO. rides from unmodified microalgae or microalgae cultured in In some embodiments, the increase in one or more of the minimal growth media under photoautotrophic conditions. above listed monosaccharides in a polysaccharide may be Non-limiting examples include polysaccharides having an from below to above detectable levels and/or by at least about increased amount of arabinose (Ara), rhamnose (Rha), fucose 60 5%, to at least about 2000%, relative to a polysaccharide (Fuc), Xylose (Xyl), glucuronic acid (GlcA), galacturonic produced from the same microalgae in the absence of genetic acid (GalA), mannose (Man), galactose (Gal), glucose (Glc), or nutritional manipulation. Therefore an increase in one or N-acetyl galactosamine (GalNAc), N-acetyl glucosamine more of the above monosaccharides, or other carbohydrates (GlcNAc), and/or N-acetylneuraminic acid (NANA), per unit listed in Tables 2 or 3, by at least about 10%, at least about mass (or per mole) of polysaccharide, relative to polysaccha 65 15%, at least about 20%, at least about 25%, at least about rides from either non-genetically modified microalgae or 30%, at least about 35%, at least about 40%, at least about microalgae cultured photoautotrophically. An increased 45%, at least about 50%, at least about 55%, at least about US 8,932,652 B2 47 48 60%, at least about 65%, at least about 70%, at least about for galactose than at least one monosaccharide selected from 75%, at least about 80%, at least about 85%, at least about the group consisting of glucose, Xylose, glucuronic acid, 90%, at least about 95%, at least about 100%, at least about mannose, and rhamnose. In additional embodiments, the 105%, at least about 110%, at least about 150%, at least about transporter has a lower K, for Xylose than at least one 200%, at least about 250%, at least about 300%, at least about monosaccharide selected from the group consisting of glu 350%, at least about 400%, at least about 450%, at least about cose, galactose, glucuronic acid, mannose, and rhamnose. In 500%, at least about 550%, at least about 600%, at least about further embodiments, the transporter has a lower K for glu 650%, at least about 700%, at least about 750%, at least about curonic acid than at least one monosaccharide selected from 800%, at least about 850%, at least about 900%, at least about the group consisting of glucose, galactose, Xylose, mannose, 1000%, at least about 1100%, at least about 1200%, at least 10 about 1300%, at least about 1400%, at least about 1500%, at and rhamnose. In yet additional embodiments, the transporter least about 1600%, at least about 1700%, at least about has a lower K for mannose than at least one monosaccharide 1800%, or at least about 1900%, or more, may be used in the selected from the group consisting of glucose, galactose, practice of the invention. Xylose, glucuronic acid, and rhamnose. In yet further embodi In cases wherein the polysaccharides from unmodified 15 ments, the transporter has a lower K, for rhamnose than at microalgae do not contain one or more of the above monosac least one monosaccharide selected from the group consisting charides, the presence of the monosaccharide in a microalgal of glucose, galactose, Xylose, glucuronic acid, and mannose. polysaccharide indicates the presence of a polysaccharide Manipulation of the concentration and identity of monosac distinct from that in unmodified microalgae. Thus using par charides provided in the culture media, combined with use of ticular strains of Porphyridium sp. and Porphyridium cruen transporters that have a different K, for different monosac tum as non-limiting examples, the invention includes modi charides, provides novel polysaccharides. These general fication of these microalgae to incorporate arabinose and/or methods can also be used in cells other than microalgae, for fucose, because polysaccharides from two strains of these example, bacteria that produce polysaccharides. species do not contain detectable amounts of these monosac In alternative embodiments, the cell is cultured in the pres charides (see Example 5 herein). In another non-limiting 25 ence of at least two monosaccharides, both of which are example, the modification of Porphyridium sp. to produce transporter by the transporter. In some cases, the two polysaccharides containing a detectable amount of glucu monosaccharides are any two selected from glucose, galac ronic acid, galacturonic acid, or N-acetylgalactosamine, or tose, Xylose, glucuronic acid, rhamnose and mannose. more thana trace amount of N-acetylglucosamine, is specifi In one non-limiting example, the method comprises pro cally included in the instant disclosure. In a further non 30 viding a transgenic cell containing a recombinant gene limiting example, the modification of Porphyridium cruen encoding a monosaccharide transporter, and culturing the cell tum to produce polysaccharides containing a detectable in the presence of at least one monosaccharide, wherein the amount of rhamnose, mannose, or N-acetylneuraminic acid, monosaccharide is transported by the transporter into the cell or more than a trace amount of N-acetyl-glucosamine, is also and is incorporated into a polysaccharide of the cell. It is specifically included in the instant disclosure. 35 pointed out that transportation of a monosaccharide from the Put more generally, the invention includes a method of media into a microalgal cell allows for the monosaccharide to producing a polysaccharide comprising culturing a microal be used as an energy source, as disclosed below, and for the gae cell in the presence of at least about 0.01 micromolarofan monosaccharide to be transported into the endoplasmic exogenously provided fixed carbon compound, wherein the reticulum (ER) by cellular transporters. In the ER, polysac compound is incorporated into the polysaccharide produced 40 charide production and glycosylation, occurs such that in the by the cell. In some embodiments, the compound is selected presence of exogenously provided monosaccharides, the from Table 2 or 3. The cells may optionally be selected from Sugar content of the microalgal polysaccharides change. the species listed in Table 1, and cultured by modification, In some aspects, the invention includes a novel microalgal using the methods disclosed herein, or the culture conditions polysaccharide, Such as from microalgae of the genus Por also listed in Table 1. 45 phyridium, comprising detectable amounts of Xylose, glu The methods may also be considered a method of produc cose, and galactose wherein the molar amount of one or more ing a glycopolymer by culturing a transgenic microalgal cell of these three monosaccharides in the polysaccharide is not in the presence of at least one monosaccharide, wherein the present in a polysaccharide of Porphyridium that is not monosaccharide is transported by the transporter into the cell genetically or nutritionally modified. An example of a non and is incorporated into a microalgal polysaccharide. 50 nutritionally and non-genetically modified Porphyridium In some embodiments, the cell is selected from Table 1, polysaccharide can be found, for example, in Jones R., Jour Such as where the cell is of the genus Porphyridium, as a nal of Cellular Comparative Physiology 60; 61-64 (1962). In non-limiting example. In some cases, the cell is selected from Some embodiments, the amount of glucose, in the polysac Porphyridium sp. and Porphyridium cruentum. Embodiments charide, is at least about 65% of the molar amount of galac include those wherein the polysaccharide is enriched for the 55 tose in the same polysaccharide. In other embodiments, glu at least one monosaccharide compared to an endogenous cose is at least about 70%, at least about 75%, at least about polysaccharide produced by a non-transgenic cell of the same 80%, at least about 85%, at least about 90%, at least about species. The monosaccharide may be selected from Arabi 95%, at least about 100%, at least about 105%, at least about nose, Fructose, Galactose, Glucose, Mannose, Xylose, Glu 110%, at least about 120%, at least about 130%, at least about curonic acid, Glucosamine, Galactosamine, Rhamnose and 60 14.0%, at least about 150%, at least about 200%, at least about N-acetylglucosamine. 250%, at least about 300%, at least about 350%, at least about These methods of the invention are facilitated by use of 400%, at least about 450%, at least about 500%, or more, of non-transgenic cell expressing a Sugar transporter, optionally the molar amount of galactose in the polysaccharide. Further wherein the transporter has a lower K, for glucose than at embodiments of the invention include those wherein the least one monosaccharide selected from the group consisting 65 amount of glucose in a microalgal polysaccharide is equal to, of galactose, Xylose, glucuronic acid, mannose, and rham or approximately equal to, the amount of galactose (such that nose. In other embodiments, the transporter has a lower K, the amount of glucose is about 100% of the amount of galac US 8,932,652 B2 49 50 tose). Moreover, the invention includes microalgal polysac phyridium, comprising detectable amounts of Xylose, glu charides wherein the amount of glucose is more than the cose, and galactose, wherein the amount of galactose in the amount of galactose. polysaccharide, is at least about 100% of the molar amount of Alternatively, the amount of glucose, in the polysaccha Xylose in the same polysaccharide. In other embodiments, ride, is less than about 65% of the molar amount of galactose 5 rhamnose is at least about 105%, at least about 110%, at least in the same polysaccharide. The invention thus provides for about 120%, at least about 130%, at least about 140%, at least polysaccharides wherein the amount of glucose is less than about 150%, at least about 200%, at least about 250%, at least about 60%, less than about 55%, less than about 50%, less about 300%, at least about 350%, at least about 400%, at least than about 45%, less than about 40%, less than about 35%, about 450%, or at least about 500%, or more, of the molar less than about 30%, less than about 25%, less than about 10 20%, less than about 15%, less than about 10%, or less than amount of mannose in the polysaccharide. Further embodi about 5% of the molar amount of galactose in the polysac ments of the invention include those wherein the amount of charide. galactose in a microalgal polysaccharide is more than the In other aspects, the invention includes a microalgal amount of xylose on a molar basis. polysaccharide, such as from microalgae of the genus Por 15 Alternatively, the amount of galactose, in the polysaccha phyridium, comprising detectable amounts of Xylose, glu ride, is less than about 55% of the molar amount of xylose in cose, galactose, mannose, and rhamnose, wherein the molar the same polysaccharide. The invention thus provides for amount of one or more of these five monosaccharides in the polysaccharides wherein the amount of galactose is less than polysaccharide is not present in a polysaccharide of non about 50%, less than about 45%, less than about 40%, less genetically modified and/or non-nutritionally modified than about 35%, less than about 30%, less than about 25%, microalgae. In some embodiments, the amount of rhamnose less than about 20%, less than about 15%, less than about in the polysaccharide is at least about 100% of the molar 10%, or less than about 5% of the molar amount of xylose in amount of mannose in the same polysaccharide. In other the polysaccharide. embodiments, rhamnose is at least about 110%, at least about In yet additional aspects, the invention includes a microal 120%, at least about 130%, at least about 140%, at least about 25 gal polysaccharide. Such as from microalgae of the genus 150%, at least about 200%, at least about 250%, at least about Porphyridium, comprising detectable amounts of xylose, glu 300%, at least about 350%, at least about 400%, at least about cose, glucuronic acid and galactose, wherein the molar 450%, or at least about 500%, or more, of the molar amount amount of one or more of these five monosaccharides in the of mannose in the polysaccharide. Further embodiments of polysaccharide is not present in a polysaccharide of unmodi the invention include those wherein the amount of rhamnose 30 in a microalgal polysaccharide is more than the amount of fied microalgae. In some embodiments, the amount of glucu mannose on a molar basis. ronic acid, in the polysaccharide, is at least about 50% of the Alternatively, the amount of rhamnose, in the polysaccha molar amount of glucose in the same polysaccharide. In other ride, is less than about 75% of the molar amount of mannose embodiments, glucuronic acid is at least about 55%, at least in the same polysaccharide. The invention thus provides for 35 about 60%, at least about 65%, at least about 70%, at least polysaccharides wherein the amount of rhamnose is less than about 75%, at least about 80%, at least about 85%, at least about 70%, less than about 65%, less than about 60%, less about 90%, at least about 95%, at least about 100%, at least than about 55%, less than about 50%, less than about 45%, about 105%, at least about 110%, at least about 120%, at least less than about 40%, less than about 35%, less than about about 130%, at least about 140%, at least about 150%, at least 30%, less than about 25%, less than about 20%, less than 40 about 200%, at least about 250%, at least about 300%, at least about 15%, less than about 10%, or less than about 5% of the about 350%, at least about 400%, at least about 450%, or at molar amount of mannose in the polysaccharide. least about 500%, or more, of the molar amount of glucose in In additional aspects, the invention includes a microalgal the polysaccharide. Further embodiments of the invention polysaccharide, such as from microalgae of the genus Por include those wherein the amount of glucuronic acid in a phyridium, comprising detectable amounts of Xylose, glu 45 microalgal polysaccharide is more than the amount of glu cose, galactose, mannose, and rhamnose, wherein the amount cose on a molar basis. of mannose, in the polysaccharide, is at least about 130% of In other embodiments, the exopolysaccharide, or cell the molar amount of rhamnose in the same polysaccharide. In homogenate polysaccharide, comprises glucose and galac other embodiments, mannose is at least about 140%, at least tose wherein the molar amount of glucose in the exopolysac about 150%, at least about 200%, at least about 250%, at least 50 charide, or cell homogenate polysaccharide, is at least about about 300%, at least about 350%, at least about 400%, at least 55% of the molar amount of galactose in the exopolysaccha about 450%, or at least about 500%, or more, of the molar ride or polysaccharide. Alternatively, the molar amount of amount of rhamnose in the polysaccharide. glucose in the exopolysaccharide, or cell homogenate Alternatively, the amount of mannose, in the polysaccha polysaccharide, is at least about 60%, at least about 65%, at ride, is equal to or less than the molar amount of rhamnose in 55 least about 70%, at least about 75%, at least about 80%, at the same polysaccharide. The invention thus provides for least about 85%, at least about 90%, or at least about 100% of polysaccharides wherein the amount of mannose is less than the molar amount of galactose in the exopolysaccharide or about 95%, less than about 90%, less than about 85%, less polysaccharide. than about 80%, less than about 75%, less than about 70%, In yet further aspects, the invention includes a microalgal less than about 65%, less than about 60%, less than about 60 polysaccharide, Such as from microalgae of the genus Por 60%, less than about 55%, less than about 50%, less than phyridium, comprising detectable amounts of Xylose, glu about 45%, less than about 40%, less than about 35%, less cose, glucuronic acid, galactose, at least one monosaccharide than about 30%, less than about 25%, less than about 20%, selected from arabinose, fucose, N-acetylgalactosamine, and less than about 15%, less than about 10%, or less than about N-acetylneuraminic acid, or any combination of two or more 5% of the molar amount of rhamnose in the polysaccharide. 65 of these four monosaccharides. In further aspects, the invention includes a microalgal In one embodiment, a method of determining the amount polysaccharide, such as from microalgae of the genus Por of phycoerythrin per dry gram of cells in a formulated skin US 8,932,652 B2 51 52 care composition is to quantify the amount of certain mol pletely, free of protein or protein as detectable by assay meth ecules known to be presentat certain levels or ranges of levels ods as described herein after treatment to remove protein. in Porphyridium cells. In further embodiments, the polysaccharide may comprise Such measurement is an indication of how many grams of a molar amount of glucose that is at least about 50%, or at dry Porphyridium biomass per gram of formulated skin care 5 least about 60%, of the molar amount of galactose. Alterna product are in a given formulation. For example, compounds tively, the molar amount of glucose in the polysaccharide is listed in FIG. 7 are known to be present in Porphyridium cells greater than the molar amount of galactose. In additional at certain ranges of levels. The amounts of compounds. Such embodiments, the polysaccharide contains less than a 0.1%, as those listed in FIG. 7, were determined by analysis of or less than a 0.01%, molar amount of at least one monosac 10 charide selected from the group consisting of arabinose, Porphyridium cell biomass grown in nitrogen-replete media rhamnose, fucose, and N-acetylglucosamine. Optionally, the under outdoor daylight conditions, i.e.: deep red cells con polysaccharide contains less than a 0.1%, or less than a taining quantities of phycoerythrin typically seen in cells 0.01%, molaramount of each of arabinose, rhamnose, fucose, grown in artificial seawater media Such as those described in and N-acetylglucosamine. Example 1. Given a certain quantity of a compound in a 15 In yet additional embodiments, a composition may com formulated skin care composition, the skilled artisan can prise a polysaccharide comprising a molar amount of glucose determine the number of dry grams of Porphyridium biomass that is at least about 30%, or at least about 40%, of the molar are present per gram of formulated skin care product. The amount of Xylose. In some cases, the polysaccharide com number of dry grams of Porphyridium cells present in the prises a molar amount of glucose between about 15.8 and composition can then be used to determine if the cells contain about 25.8%; and a molar amount of xylose that is between less than or more than a certain amount of phycoerythrin per about 37.5 and about 45.5%. Alternatively, the polysaccha dry gram of cells. ride comprises a molaramount of glucose between about 17.8 IV Cosmeceutical Compositions and Topical Application and about 23.8%; and a molar amount of xylose that is A. General between about 39.5 and about 43.5%. Compositions, comprising polysaccharides, whole cell 25 In yet further embodiments, the polysaccharides are Sul extracts, or mixtures of polysaccharides and whole cell fated exopolysaccharides containing at least 3.0% about Sul extracts, are provided for topical application or non-systemic fur, at least about 3.5% sulfur, at least about 4.0% sulfur, at administration. The polysaccharide may be any one or more least about 4.5% sulfur, at least about 4.6% sulfur, at least of the microalgal polysaccharides disclosed herein, including about 4.75% sulfur, at least about 5.0% sulfur, at least about those produced by a species, or a combination of two or more 30 5.25% sulfur, at least about 5.5% sulfur, at least about 5.75% species, in Table 1. Similarly, a whole cell extract may be that sulfur, at least about 6.0% sulfur, at least about 6.25% sulfur, prepared from a microalgal species, or a combination of two at least about 6.5% sulfur, at least about 6.75% sulfur, or at or more species, in Table 1. In some embodiments, polysac least about 7.0% sulfur by weight of the polysaccharide. The charides, such as exopolysaccharides, and cell extracts from amount or level of Sulfation in the polysaccharides may be microalgae of the genus Porphyridium are used in the practice 35 analyzed and compared to the amount of Sulfates used to of the invention. A composition of the invention may com culture the microalgae. Thus the amount or level of sulfation prise from between about 0.001% and about 100%, about in the polysaccharides of cells grown at about 100 mM, about 0.01% and about 90%, about 0.1% and about 80%, about 1% 200 mM, about 300 mM, about 400 mM, about 500 mM, and about 70%, about 2% and about 60%, about 4% and about about 600 mM, or about 700 mM or higher, sulfate (SO) 50%, about 6% and about 40%, about 7% and about 30%, 40 may be determined by routine and repetitive methods dis about 8% and about 20%, or about 10% polysaccharide, cell closed herein. The amount or level of sulfur by weight in the extract, by weight. polysaccharides of a sample of cells or cell material may In other embodiments, the composition comprises a carrier determined without knowledge of the amount of sulfate used Suitable for topical administration and/or a preservative Suit to culture the cells. able for topical administration; and Rhodophyte cells, option 45 As a further alternative, a composition for topical applica ally of the genus Porphyridium. The cells may contain tion to human skin may comprise microalgal cells. The cells reduced amounts of the red pigmentation by preparation may be those of genus Porphyridium or any other species or methods described herein. In some cases, an aqueous extract strain as disclosed herein. Optionally, the composition further of a composition comprising the Porphyridium cells contains comprising a carrier and/or preservative Suitable for topical no more than 75% to no more than about 1% of the absor 50 administration as described herein. In alternative embodi bance per gram at 545 nm of a second composition formu ments, the cells are homogenized (such as by methods lated in identical fashion except containing cells of the same described herein) to generate or form a microalgal cell homo species of Porphyridium cells that were grown in a photo genate. In some cases, the cells or homogenate thereof, and bioreactor in ATCC 1495 ASW media in the presence of 50 therefore the composition, is essentially free of red and/or microeinsteins of light per square meter per second. In further 55 green coloration. Optionally, the cells or homogenate thereof, embodiments, the carrier is suitable for topical administration and therefore the composition, is completely free of red col to humans, such as to human skin or a skin tissue. oration. Thus in some embodiments, the cells (and therefore In alternative embodiments, a composition for application the homogenate thereof) contains less than about 15, less than to human skin may comprise a polysaccharide isolated from about 10, less than about 5, less than about 2, less than about cells of the genus Porphyridium. Such a composition may 60 1, less than about 0.5, or less than about 0.1 milligrams of further comprise a carrier and/or preservative suitable for phycoerythrin per dry gram of cells. topical administration as described herein. In some cases, the Alternatively, the cells (and therefore a homogenate polysaccharide of the composition contains no more than thereof) contains a Sulfated polysaccharide having an amount about 10% protein by weight. In other embodiments, the of sulfur by weight of at least 3.0%, at least 3.5%, at least polysaccharide contains no more than about 5%, no more 65 4.0%, at least 4.5%, at least 4.6%, at least 4.75%, at least than about 2%, or no more than about 1% protein by weight. 5.0%, at least 5.25%, at least 5.5%, at least 5.75%, at least Of course the polysaccharide may also be essentially, or com 6.0%, at least 6.25%, at least 6.5%, at least 6.75%, or at least US 8,932,652 B2 53 54 7.0% sulfur by weight of the polysaccharide as described 24(12):2083-96; Int J. Pharm. 2003 Nov. 28:267(1-2):13-25; herein. In additional embodiments, the microalgal cell homo Med Biol Eng Comput. 1998 January: 36(1): 129-34 and genate contains at least two, at least three, at least five, at least Reprod Fertil Dev. 2002: 14(5-6):307-14. A representative ten, or at least twenty times the amount of solvent-available example is chemical cross-linking to a pharmaceutically or polysaccharide present in a quantity of unhomogenized cells cosmetically acceptable insoluble Solid phase material. Such needed to generate the microalgal cell homogenate. as a polymer, microbead, or nanobead. The insoluble material In further embodiments, the disclosed invention includes a need not precipitate when in a solution but includes a material composition comprising particulate polysaccharides, such as that remains in Suspension when in solution. Dehydration or microbeads or nanobeads comprising a disclosed polysaccha precipitation with alcohol may be practiced with any alcohol ride. In some embodiments the polysaccharide particles are 10 Suitable for pharmaceutical or cosmetic use. Non-limiting referred to as Marine NanobeadsTM. The composition may be examples include ethanol or a fatty alcohol Such as cetyl, for improving the appearance of skin, Such as human skin. Stearyl, cetearyl, or lanolin alcohol. A non-limiting method of The polysaccharides may have any level of sulfation microencapsulating a cosmetic is described in U.S. Pat. No. described herein. The composition may be sterile and/or non 4,752,496. pyrogenic and optionally substantially free of endotoxins 15 The use of a disclosed method of the invention also and/or proteins. In other embodiments, the composition fur includes milling of dried polysaccharide material (such as a ther comprises hyaluronic acid or another agent Suitable or film) into particles by any suitable method. Non-limiting desirable for treatment of skin. Non-limiting examples of examples of Such methods are disclosed herein, and they Such an agent include aloe Vera, urea, alpha hydroxyl acid, produce particles with an average size that may range Vitamin E, glycyrrhizinic acid, methylsulfonylmethane between about 400 and about 0.1 microns. (MSM), and collagen. In some embodiments, the composition comprises In Some embodiments, the composition comprises an algal polysaccharide particles that increase in Volume on contact polysaccharide, wherein the polysaccharide: (a) has been with water compared to their anhydrous or partially hydrated made completely or partially insoluble in water through dry Volume. In some embodiments, the particles increase in Vol ing; and (b) has been homogenized or otherwise milled or 25 ume by an amount selected from at least about 5%, at least disrupted to generate particles. about 25%, at least about 50%, at least about 100%, at least The polysaccharide may of course be that of a variety of about 200%, at least about 300%, by at least about 500%, at microalgal cells, such as those described in Table 1 and those least about 1000%, or at least about 5000%. of the genus Porphyridium. In some cases, the polysaccharide In some embodiments, the polysaccharide of the method is is contained in a non-aqueous material. As non-limiting 30 associated with a fusion protein as described herein. In some examples, the material may be contained in an oil suitable for cases, the fusion protein comprises a first protein with at least topical administration, with hexadecanoic acid or oil that is 60% amino acid identity with the protein of SEQID NO: 15, contained in an emulsion as representative examples. The and a second protein. Alternatively, the polysaccharide of the composition may also comprise a carrier and/or preservative method contains an amount of Sulfur by weight from at least suitable for topical administration. Of course the composition 35 about 3.0% sulfur to at least about 7.0% sulfur by weight as may also be substantially free of endotoxins and/or protein as described herein, and optionally is also associated with a well as sterile and/or non-pyrogenic. In further embodiments, fusion protein. An example of an expression vector for the polysaccharide is encapsulated by a timed-release coat expression in Porphyridium of a polysaccharide binding pro ing, Such as one Suitable for topical application to human skin. tein:superoxide dismutase fusion can be found in SEQ ID Optionally, the composition is prepared by a manufactur 40 NO:36. In some embodiments a spacer of 1-15 amino acids ing or preparation method as described herein, such as a is placed between the glycoprotein and second protein to method disclosed in the following Methods of Formulation enable flexibility between the two proteins. Expression of a section. So in some cases, the composition comprises a fusion protein, wherein the second heterologous protein is a polysaccharide that is partially or completely insoluble in dimerizing or multimerizing protein (Such as Superoxide dis water, Such as by heating an aqueous Suspension of the 45 mutase) can be advantageous when a higher viscosity or polysaccharide thereby removing water from the Suspension. gelling property of the polysaccharide is desired because the The polysaccharide particulates may be partially soluble Such dimmers serve to crosslink the polysaccharide. The revers that they are less than about 70%, less than about 60%, less ibility of the crosslinking is in part dictated by the strength of than about 50%, less than about 40%, less than about 30%, the dimerization in such fusion proteins provided by the less than about 20%, less than about 10%, less than about 5%, 50 invention. or less than about 2% soluble in water. Topical compositions are usually formulated with a carrier, In other embodiments, the polysaccharide has been made Such as in an ointment or a cream, and may optionally include partially or completely insoluble by a method selected from a fragrance. One non-limiting class oftopical compositions is the group consisting of chemical cross-linking, chemical that of cosmeceuticals. Other non-limiting examples of topi dehydration through displacement of bound water by an alco 55 cal formulations include gels, solutions, impregnated ban hol, precipitation from Solution using an alcohol or a ketone dages, liposomes, or biodegradable microcapsules as well as or pH, and coating of particles by microencapsulation. Non lotions, sprays, aerosols, Suspensions, dusting powder, limiting examples of these methods are known to the skilled impregnated bandages and dressings, biodegradable poly person and may be used in the practice of the invention. For mers, and artificial skin. Another non-limiting example of a examples, see Biomacromolecules. 2005 Nov.-Dec. 6(6): 60 topical formulation is that of an ophthalmic preparation. Car 3202-8; Arterioscler Thromb Vase Biol. 2004 March; 24(3): riers for topical administration of the compounds of this 613-7: J Biomed Mater Res. 2001 Sep. 15: 56(4):478-86: invention include, but are not limited to, mineral oil, liquid Dalton Trans. 2004 Sep. 7: (17):2621-34. Epub 2004 Jul. 28: petroleum, white petroleum, propylene glycol, polyoxyeth Biomacromolecules. 2004 January-February: 5(1): 126-36; ylene polyoxypropylene compound, emulsifying wax and Contraception. 2002 August: 66(2): 137-40; Biomacromol 65 water. Alternatively, the composition can be formulated with ecules. 2006 May: 7(5):1471-80; Biopolymers. 1999 Sep a Suitable lotion or cream containing the active compound tember; 50(3):227-37; Biomaterials. 2003 May; suspended or dissolved in a carrier. Suitable carriers include, US 8,932,652 B2 55 56 but are not limited to, mineral oil, Sorbitan monostearate, antigen from a pathogen selected from HIV. Herpes Simplex polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyl Virus, gonorrhea, Chlamydia, Human Papillomavirus, and dodecanol, benzyl alcohol and water. Trichomoniasis. In some embodiments, the antibody is a In some embodiments, the polysaccharides contain fucose humanized antibody. moieties. In other embodiments, the polysaccharides are Sul B. Methods of Formulation fated, such as exopolysaccharides from microalgae of the Polysaccharide compositions for topical application can be genus Porphyridium. In some embodiments, the polysaccha formulated by first preparing a purified preparation of rides will be those from a Porphyridium species, such as one polysaccharide. As a non-limiting example, the polysaccha that has been Subject to genetic and/or nutritional manipula ride from aqueous growth media is precipitated with an alco tion to produce polysaccharides with altered monosaccharide 10 hol, resuspended in a dilute buffer, and mixed with a carrier content and/or altered sulfation. Suitable for application to human skin or mucosal tissue, In additional embodiments, a composition of the invention including the vaginal canal. Alternatively, the polysaccharide comprises a microalgal cell homogenate and a topical carrier. can be purified from growth media and concentrated by tan In some embodiments, the homogenate may be that of a gential flow filtration or other filtration methods, and formu species listed in Table 1 or may be material produced by a 15 lated as described above. Intracellular polysaccharides can be species in the table. also formulated in a similar or identical manner after purifi In further embodiments, a composition comprising puri cation from other cellular components. fied microalgal polysaccharide and a carrier Suitable for topi As a non-limiting example, the invention includes a cal administration also contains a fusion (or chimeric) protein method of formulating a cosmeceutical composition, said associated with the polysaccharide. In some embodiments, method comprising culturing microalgal cells in Suspension the fusion protein comprises a first protein, or polypeptide under conditions to allow cell division; separating the region, with at least about 60% amino acid identity with the microalgal cells from culture media, wherein the culture protein of SEQ ID NO: 15. In other embodiments, the first media contains exopolysaccharide molecules produced by protein has at least about 70%, at least about 75%, at least the microalgal cells; separating the exopolysaccharide mol about 80%, at least about 85%, at least about 90%, at least 25 ecules from other molecules present in the culture media; about 95%, or at least about 98%, or higher, amino acid homogenizing the microalgal cells; and adding the separated identity with the sequence of SEQID NO:15. Preferably the exopolysaccharide molecules to the cells before, during, or fusion protein binds to a Sulfated exopolysaccharide from a after homogenization. In some embodiments, the microalgal cell of the genus Porphyridium. It is preferable that the bind cells are from the genus Porphyridium. ing of the fusion protein to the polysaccharide be selective 30 In other embodiments, the invention includes a method of and high affinity, though Such is not required to practice the manufacturing a composition comprising particles, the invention. method comprising isolating a polysaccharide from microal The fusion protein may comprise a second protein, or gae; drying an aqueous Suspension of the polysaccharide to a polypeptide region, with a homogenous or heterologous solid film wherein at least some proportion of the film has sequence. Non-limiting examples of the second protein 35 been made completely or partially insoluble in water, homog include an antibody, an enzyme, or a structural protein of skin enizing or otherwise milling or disrupting the film into par or a skin tissue, such as that of a human being. In optional ticles; and formulating the particles into a non-aqueous mate embodiments, the enzyme is Superoxide dismutase, Such as rial. that has at least about 60% amino acid identity with the The method may of course be practiced with a variety of sequence of SEQID NO: 12, SEQ ID NO: 13, or a protein 40 microalgal cells, such as those described in Table 1 and those from Table 21 and exhibit superoxide dismutase activity as of the genus Porphyridium. non-limiting examples. In some embodiments, the SuperoX As described herein, the resulting composition may be for ide dismutase has at least about 70%, at least about 75%, at improving the appearance of skin, Such as human skin. In least about 80%, at least about 85%, at least about 90%, at Some embodiments, the formulating may be into the oil phase least about 95%, or at least about 98%, or higher, amino acid 45 of an oil-in-water emulsion. In other embodiments, the non identity with the sequence of SEQID NO:12 or 13. In other aqueous material is an oil suitable for topical administration, embodiments, the second protein is a structural skin protein with hexadecanoic acid and oil that is contained in an emul selected from the group consisting of elastin and a collagen sion as non-limiting examples. In further embodiments, the chain, Such as that of human skin. Sequences encoding elastin method further comprises formulating the particles into a and a chain of collagen are known to the skilled person and 50 carrier and/or preservative suitable for topical administration. may be incorporated into a fusion protein via routine meth The resulting composition may also be substantially free of ods. Examples of such human skin proteins are also disclosed endotoxins and/or protein. In many embodiments, the com herein in the sequence listing. Assays for Superoxide dismu position is also made sterile and/or non-pyrogenic. Alterna tase activity are well known in the art. For examples see Song tively, the method further comprises formulating hyaluronic et al., Clin Sci (Lond). 2007 Jan. 8: Epub ahead of print, 55 acid into the composition. Oxidative stress, antioxidant status and DNA damage in In other embodiments, the polysaccharide after the drying patients with impaired glucose regulation and newly-diag step is partially or completely insoluble in water. Optionally, nosed Type II diabetes; and Liu et al., Phytomedicine. 2006 the polysaccharide after the drying step is soluble in water at Dec. 15 Protection of PC12 cells from hydrogen peroxide a percentage selected from the list consisting of less than induced cytotoxicity by Salvianolic acid B, a new compound 60 about 70%, less than about 60%, less than about 50%, less isolated from Radix Salviae miltiorrhizae. The presence of than about 40%, less than about 30%, less than about 20%, any exogenous or endogenous protein expressed in microal less than about 10%, less than about 5%, and less than about gae can be assayed for example, using well known methods 2%. Such as western blotting and ELISA assays. Embodiments of the drying step include drying performed In other embodiments, the second protein is an antibody. 65 at between about 40 and about 180°C., such as between about Non-limiting examples of antibodies for use in this aspect of 80 and about 170, or between about 100 and about 160, the invention include an antibody that selectively binds to an between about 125 and about 155, between about 135 and US 8,932,652 B2 57 58 about 152, between about 140 and about 150, or between Cells can also be dried and ground using means such as about 145 and about 148° C. as non-limiting examples. mortar and pestle, colloid milling, ball milling, or other physi Optionally, the drying is performed in two steps, wherein the cal method of breaking cell walls. first step comprises heating the Suspension of the polysaccha After cell disruption, cell homogenate can be formulated ride to no more than about 60°C. for a first period of time to 5 with carrier and fragrance as described above for polysaccha produce a solid film followed by heating the solid film for a rides. second period of time to no more than about 160° C. In The compositions according to the present invention can alternative embodiments, the first and second steps comprise also be used as hair treating agents such as hairdressings (e.g., heating to no more than about 80 and no more than about 150, hair creams, hair sprays, hair tonics, hair gels, hair lotions, 10 hair oils, hair essences, hair waters, hair waxes, and hair or to approximately 100 and no more than 148°C., respec mousses), shampoos, finishing rinses, hair treatments, hair tively. In some embodiments, the Suspension of the polysac creams, hair mousses, hair setting lotions, hair colors, hair charide is heated during the first period oftime in the presence dyes (e.g., hair colors, one-part hair dyes, and two-part hair ofair to produce a solid film and the solid film is heated during dyes), perm solutions (e.g., permanent wave solutions, hair the second period of time in at least a partial vacuum or 15 straightening Solutions, and permanent wave holding agents), otherwise under reduced pressure. blood flow enhancers, Scalp lotions, and anti-hair loss agents. After the drying step, milling may be by any Suitable Other application of the compositions according to the method. Non-limiting examples include a method selected present invention include, for example, skin care cosmetics from the list consisting of jet milling, ball milling. Retsch R) Such as toners, serums, whitening toners, milky lotions, whit milling, and milling in a QuadroR device. The resulting par ening milky lotions, creams, whitening creams, ointments, ticles of the composition may have an average size between whitening ointments, lotions, whitening lotions, oils, facial about 400 and about 0.1 microns. In some embodiments, the packs. Furthermore, still other applications of the composi particles of the composition have an average size between tions according to the present invention includes, for about 100 and about 0.1 microns, between about 50 and about example, makeup cosmetics Such as foundations, liquid foun 0.1 microns, between about 10 and about 0.1 microns, 25 dations, lipsticks, lip glosses, eye shadows, powders, face between about 10 and about 0.5 microns, or between about 5 powders, blushers, eye shadows, eye liners, mascaras, and and about 0.5 microns. eyebrow pencils. Other applications of the compositions In Some embodiments, the polysaccharide of the method is according to the present invention include, for example, skin associated with a fusion protein as described herein. In some cleaners such as Soap, cleansing creams, cleansing lotions, cases, the fusion protein comprises a first protein with at least 30 cleansing milks, cosmetic compositions, facial washes, and 60% amino acid identity with the protein of SEQID NO: 15, body shampoos. Moreover, another application of the com and a second protein. Alternatively, the polysaccharide of the positions according to the present invention include finishing method contains an amount of Sulfur by weight from at least cosmetics such as manicures. Other applications of the com about 3.0% sulfur to at least about 7.0% sulfur by weight as positions according to the present invention include, for described herein, and in some embodiments is associated 35 example, cosmetic compositions in the form of bath agents, with a fusion protein. patches, perfumes, toothpastes, tooth washes, and mouth Examples of polysaccharides, both secreted and intracel washes. lular, that are suitable for formulation with a carrier for topical C. Co-Administered Compositions application are listed in Table 1. Topical compositions can comprise a portion of a complete In further embodiments, polysaccharide is associated with 40 composition sold as a single unit. Other portions of the com a fusion (or chimeric) protein comprising a first protein (or plete compositions can comprise an oral Supplement intended polypeptide region) with at least about 60% amino acid iden for administration as part of a regime for altering skin appear tity with the protein of SEQID NO: 15. In some cases, the first ance. Because the top layers of the skin contain dead cells, protein has at least about 70%, at least about 75%, at least nutrients delivered via capillaries cannot reach the outer lay about 80%, at least about 85%, at least about 90%, at least 45 ers of cells. The outer layers of cells must be provided with about 95%, or at least about 98%, or higher, amino acid nutrients though topical administration. However, topical identity with the sequence of SEQID NO:15. administration is not always an effective method of providing The fusion protein may comprise a second protein, or nutrients to deep layers of skin that contain living cells. The polypeptide region, with a homogenous or heterologous compositions provided herein comprise both topical compo sequence. One non-limiting example of the second protein is 50 sitions that contain algal polysaccharides and/or cellular a Superoxide dismutase enzyme. extracts as well as oral compositions comprising nutraceuti Examples of carriers Suitable for formulating polysaccha cal molecules Such as purified polysaccharides, whole cell ride are described above. Ratios of homogenate:carrier are extracts, carotenoids, polyunsaturated fatty acids, and other typically in the range of about 0.001:1 to about 1:1 (volume: molecules that are delivered to the skin via capillaries. The Volume), although the invention comprises ratios outside of 55 combined effect of the topical and oral administration of these this range, such as, but not limited to, about 0.01:1 and about molecules and extracts provides a benefit to skinhealth that is O.1:1 additive or synergistic compared to the use of only a topical or Microalgal cellular extracts can also be formulated for only an orally delivered product. topical administration. It is preferable but not necessary that Examples of the topical components of the composition the cells are physically or chemically disrupted as part of the 60 include exopolysaccharide from Porphyridium cruentum, formulation process. For example, cells can be centrifuged Porphyridium sp., list others. Other components of the topical from culture, washed with a buffer such as 1.0 mM phosphate composition can include polysaccharides and/or cell extracts buffered saline, pH 7.4, and sonicated. Preferably the cells are from species listed in Table I. sonicated until the cell walls have been substantially dis Cellular extracts for topical administration can also include rupted, as can be determined under a microscope. For 65 cellular homogenates from microalgae that have been geneti example, Porphyridium sp. cells can be Sonicated using a cally engineered. For example, homogenates of Porphy Misonix sonicator as described in Example 3. ridium sp. that have been engineered to express an exogenous US 8,932,652 B2 59 60 gene encoding Superoxide dismutase can be formulated for capable of being expressed in the cells or be a sequence which topical administration. Other genes that can be expressed increases expression of one or more endogenous microalgal include carotenoid biosynthesis enzymes and polyunsatu gene product. rated fatty acid biosynthesis enzymes. In a preferred embodiment, at the topical composition and Examples of compositions for oral administration include the oral composition both contain at least one molecule in one or more of the following: DHA, EPA, ARA, lineoileic common. For example, the topical composition contains acid, lutein, lycopene, beta carotene, braunixanthin, Zeaxan homogenate of Porphyridium cells that contain zeaxanthin, thin, astaxanthin, linoleic acid, alpha carotene, Vitamin C and and the oral composition contains zeaxanthin. In another Superoxide dismutase. Compositions for oral administration embodiment, the topical composition contains homogenate 10 of Porphyridium cells that contain polysaccharide, and the usually include a carrier such as those described above. Oral oral composition contains polysaccharide purified from Por compositions can be formulated in tablet or capsule form. phyridium culture media. Oral compositions can also be formulated in an ingestible Some of the compositions described herein are packaged form Such as a food, tea, liquid, etc. Oral compositions can, for sale as a single unit. For example, a unit for sale comprises for example, comprise at least 50 microgram, at least 100 15 a first container holding a composition for topical adminis microgram, at least 50 milligrams, at least 100 milligrams, at tration, a second container holding individual doses of a com least 500 milligrams, and at least one gram of a small mol position for oral administration, and optionally, directions for ecule Such as a carotenoids or a polyunsaturated fatty acid. co-administration of the topical and oral composition. In another aspect, the invention includes orally adminis Some embodiments of the invention include a combination tered nutraceutical compositions comprising one or more product comprising 1) a first composition comprising a micro polysaccharides, or microalgal cell extract or homogenate, of algal extract and a carrier Suitable for topical application to the invention. A nutraceutical composition serves as a nutri skin; and 2) a second composition comprising at least one tional Supplement upon consumption. In other embodiments, compound and a carrier Suitable for human consumption; a nutraceutical may be bioactive and serve to affect, alter, or wherein the first and second compositions are packaged for regulate a bioactivity of an organism. 25 sale as a single unit. Thus the invention includes co-packag A nutraceutical may be in the form of a solid or liquid ing of the two compositions, optionally with a instructions formulation. In some embodiments, a solid formulation and/or a label indicating the identity of the contents and/or includes a capsule or tablet formulation as described above. their proper use. In other embodiments, a Solid nutraceutical may simply be a Other combination products are including in the invention. dried microalgal extract or homogenate, as well as dried 30 In some embodiments, the first composition may be a topical polysaccharides perse. In liquid formulations, the invention formulation or non-systemic formulation, optionally a cos includes suspensions, as well as aqueous solutions, of meceutical, as described herein. Preferably, the first compo polysaccharides, extracts, or homogenates. In some embodi sition comprises a carrier Suitable for topical application to ments the nutraceutical is derived from microalgae, while in skin, Such as human skin. Non-limiting examples of the sec other embodiments the nutraceutical is derived from other 35 ond composition include a food composition or nutraceutical Sources such as, for example, plants, plant extracts, and as described herein. Preferably, the second composition com chemically synthesized molecules. In a preferred embodi prises at least one carrier Suitable for human consumption, ment a topical composition and an oral composition contain Such as that present in a food product or composition. Com at least one molecule in common. bination products of the invention may be packaged sepa The methods of the invention include a method of produc 40 rately for Subsequent use together by a user or packaged ing a nutraceutical composition. Such a method may com together to facilitate purchase and use by a consumer. Pack prise drying a microalgal cell homogenate or cell extract. The aging of the first and second compositions may be for sale as homogenate may be produced by disruption of microalgae a single unit. which has been separated from culture media used to propa D. Methods of Cosmetic Enhancement gate (or culture) the microalgae. Thus in one non-limiting 45 In a further aspect, the invention includes a method to example, a method of the invention comprises culturing red cosmetically enhance skin or its appearance or texture. In microalgae, separating the microalgae from culture media; Some cases, the enhancement is due to increased or improved disrupting the microalgae to produce a homogenate; and dry skin elasticity. The skin may be that of a human being, such as ing the homogenate. In similar embodiments, a method of the the skin of the face, hands, feet, or other parts of the human invention may comprise drying one or more polysaccharides 50 body. In other embodiments, the enhancement may be in the produced by the microalgae. appearance or texture of human lips. The method may com In some embodiments, a method of the invention com prise administration of a polysaccharide composition Suitable prises drying by tray drying, spin drying, rotary drying, spin for injection into skin or lip tissue to improve the appearance flash drying, or lyophilization. In other embodiments, meth thereof. The composition may be any as described herein ods of the invention comprise disruption of microalgae by a 55 suitable for the method of administration or application. In method selected from pressure disruption, Sonication, and Some embodiments, the injection is made to alleviate or ball milling eliminate wrinkles. In other embodiments, the treatment In additional embodiments, a method of the invention fur reduces the visible signs of aging and/or wrinkles. ther comprises formulation of the homogenate, extract, or As known to the skilled person, human skin, as it ages, polysaccharides with a carrier Suitable for human consump 60 gradually loses skin components that keep skin pliant and tion. As described herein, the formulation may be that of youthful-looking. The skin components include collagen, tableting or encapsulation of the homogenate or extract. elastin, and hyaluronic acid, which have been the subject of In further embodiments, the methods comprise the use of interest and use to improve the appearance of aging skin. microalgal homogenates, extracts, or polysaccharides The invention includes compositions of microalgal wherein the cells contain an exogenous nucleic acid 65 polysaccharides, microalgal cell extracts, and microalgal cell sequence. Such as in the case of modified cells described homogenates for use in the same manner as collagen and herein. The exogenous sequence may encode a gene product hyaluronic acid. In some embodiments, the polysaccharides US 8,932,652 B2 61 62 will be those of from a Porphyridium species, such as one that The invention also includes a method to stimulate procol has been Subject to genetic and/or nutritional manipulation to lagen and/or collagen synthesis or production in a cell. Such produce polysaccharides with altered monosaccharide con as a human fibroblast, by contacting the cell with a disclosed tent and/or altered sulfation. In some embodiments, the polysaccharide. In some embodiments, the cell is in the skin polysaccharides are formulated as a fluid, optionally elastic 5 of a human Subject and the contacting comprises administer and/or viscous, Suitable for injection. The compositions may ing the polysaccharide to the Subject. The administering may be used as injectable dermal fillers as one non-limiting comprise injection of the polysaccharide, or a polysaccharide example. The injections may be made into skin to fill-out containing composition of the invention, to the skin or a skin facial lines and wrinkles. In other embodiments, the injec tissue and/or to the lips or a lip tissue. The amount of polysac tions may be used for lip enhancement. These applications of 10 charide administered may be any that is sufficient or effective polysaccharides are non-limiting examples of non-pharma to stimulate procollagen or collagen synthesis to a level desired by a skilled person, Such as an increase of at least cological therapeutic methods of the invention. about 5%, 10%, about 20%, or about 30% or higher than that In further embodiments, the microalgal polysaccharides, observed in the absence of polysaccharide. In a related man cell extracts, and cell homogenates of the invention may be 15 ner, the polysaccharide may be used to inhibit collagenase co-formulated with collagen and/or hyaluronic acid (Such as activity. The inhibition may be sufficient to result in an the Restylane(R) and Hylaform R) products) and injected into increase of procollagen or collagen levels as described above. facial tissue. Non-limiting examples of Such tissue include Additionally, the invention includes a method to stimulate under the skin in areas of wrinkles and the lips. In a preferred elastin synthesis or production in a cell. Such as a fibroblast, embodiment, the polysaccharide is substantially free of pro by contacting the cell with a disclosed polysaccharide. In a tein. The injections may be repeated as deemed appropriate related manner, the polysaccharide may also inhibit elastase by the skilled practitioner, such as with a periodicity of about activity produced by a cell. Such as, but not limited to, a three, about four, about six, about nine, or about twelve fibroblast. In some embodiments, the cell is in the skin of a months. In another preferred embodiment, a hyaluronic acid human Subject and the contacting comprises administering material is mixed with a polysaccharide from the genus Por 25 the polysaccharide to the Subject. The administering may phyridium prior to co-administration. The invention in this comprise injection of the polysaccharide, or a polysaccharide particular embodiment provides longer half-life to the hyalu containing composition of the invention, to the skin or a skin ronic acid due to the potent inhibition of hyaluronidase by tissue. The amount of polysaccharide administered may be polysaccharides isolated from microalgae from the genus any that is sufficient or effective to stimulate elastin synthesis Porphyridium. This allows for less injections to a patient. 30 to a level desired by a skilled person, such as an increase of at Preferably the polysaccharide from the genus Porphyridium least about 50%, 100%, about 200%, or about 300% or higher is at least substantially free of protein. Preferably the mixture than that observed in the absence of polysaccharide. In a of polysaccharide from the genus Porphyridium and hyalu preferred embodiment the polysaccharide stimulating elastin ronic acid is sterile. secretion contains at least 5.0% sulfur by weight. Similarly, Thus the invention includes a method of cosmetic enhance 35 the polysaccharide may decrease elastase activity by about ment comprising injecting a polysaccharide produced by 10%, about 20%, about 30%, about 40%, about 50%, or about microalgae into mammalian skin. The injection may be of an 60% or higher than that observed in the absence of polysac effective amount to produce a cosmetic improvement, such as charide. decreased wrinkling or decreased appearance of wrinkles as The invention further includes the use of the disclosed non-limiting examples. Alternatively, the injection may be of 40 polysaccharides based on their observed anti-oxidantactivity. an amount which produces relief in combination with a series Thus the invention includes a method of providing anti-oxi of additional injections. In some methods, the polysaccharide dant activity to skin or a skin tissue. Such as that of a human is produced by a microalgal species, or two or more species, Subject, by administering a polysaccharide. In some embodi listed in Table 1. In one non-limiting example, the microalgal ments, the method inhibits reactive oxygen species (ROS) species is of the genus Porphyridium and the polysaccharide 45 formation and/or activity in the skin. The invention thus is substantially free of protein. includes a method to prevent or treat a disease or unwanted The invention further includes a method to inhibit hyalu condition associated with ROS or oxidative stress. Non-lim ronidase activity comprising contacting the hyaluronidase iting examples of Such a disease or unwanted condition with a disclosed polysaccharide. In some embodiments, the include reducing inflammation or irritation of human skin or hyaluronidase activity is in the skin or a skin tissue of a human 50 lips. In some embodiments, the polysaccharide composition Subject and the contacting comprises administering the comprises one or more other agents or compounds with anti polysaccharide to the Subject. The administering may com oxidant activity. Non-limiting examples of other agents prise injection of the polysaccharide, or a polysaccharide include vitamin A (retinyl palmitate), Vitamin C (such as one containing composition of the invention, to the skin or skin or more of ascorbyl palmitate, Sodium ascorbyl palmitate, tissue and/or to the lips or a lip tissue. The amount of polysac 55 and tetrahexyldecylascorbate), vitamin D (cholecalcipherol), charide administered may be any that is sufficient or effective Vitamin E (Such as tocopheryl acetate and tocopherol/D-alpha to inhibit hyaluronidase activity to a level as desired by a tocopherol), alpha lipoic acid, coenzyme, L-selenomethion skilled person. The level of reduction in hyaluronidase activ ine, and beta glucan. ity may be determined by routine methods, including a com In a related manner, a polysaccharide is used based on its parative method wherein the activity in the presence of 60 anti-inflammatory in skin or a skin tissue. In some embodi polysaccharide is compared to the activity in the absence ments, the method inhibits polymorphonuclear (PMN) leu thereof. Thus the amount of reduction may be at least about kocytes in chemotaxis, such as to sites of inflammation in 10%, about 20%, about 30%, about 40%, about 50%, about skin. The level of inhibition may be about 10%, about 20%, 60%, about 70%, about 80%, or about 90% or higher than that about 30%, about 40%, or about 50% or more than that seen observed in the absence of polysaccharide. In a preferred 65 in the absence of polysaccharide. In other embodiments, the embodiment the polysaccharide used to inhibit hyaluronidase method inhibits the synthesis or release of a pro-inflamma is from a species of the genus Porphyridium. tory cytokine, such as interferon-gamma or interleukin-1- US 8,932,652 B2 63 64 alpha. With interferon-gamma as an example, the inhibition V Gene Expression in Microalgae may be at least about 10%, about 20%, about 30%, about Genes can be expressed in microalgae by providing, for 40%, about 50%, about 60%, about 70%, about 80%, or about example, coding sequences in operable linkage with promot 90% or more than that observed in the absence of polysac CS. charide. With interleukin-1-alpha as an example, the inhibi An exemplary vector design for expression of a gene in tion may be at least about 10%, about 20%, about 30%, about microalgae contains a first gene in operable linkage with a 40%, about 50%, about 60%, about 70%, or about 80% or promoteractive in algae, the first gene encoding a protein that more than that observed in the absence of polysaccharide. In imparts resistance to an antibiotic or herbicide. Optionally the further embodiments, the method inhibits proliferation of first gene is followed by a 3' untranslated sequence containing peripheral blood mononuclear cells, including lymphocytes, 10 monocytes, and macrophages. The level of inhibition may be a polyadenylation signal. The vector may also contain a sec about 10%, about 20%, about 30%, about 40%, about 50%, ond promoter active in algae in operable linkage with a sec about 60%, about 70%, or about 80% or more than that ond gene. The second gene can encode any protein, for observed in the absence of polysaccharide. example an enzyme that produces Small molecules or a mam The above described methods may be individually part of a 15 malian growth hormone that can be advantageously present in method to reduce the signs of aging or reduce the appearance a nutraceutical. ofaging in human skinas described herein. The methods may It is preferable to use codon-optimized cDNAs: for meth also be based upon the insight that the microalgal biomass and ods of recoding genes for expression in microalgae, see for polysaccharides of the invention also reduce the effects of UV example US patent application 20040209256. light or radiation. In some embodiments, the polysaccharide It has been shown that many promoters in expression vec reduces thymidine dimer formation in DNA caused by expo tors are active in algae, including both promoters that are sure to UVB irradiation. The reduction may be at least about endogenous to the algae being transformed algae as well as 10%, about 20%, about 30%, about 40%, about 50%, about promoters that are not endogenous to the algae being trans 60%, about 70%, or about 80% or more than that observed in formed (i.e.: promoters from other algae, promoters from the absence of polysaccharide. 25 plants, and promoters from plant viruses or algae viruses). In a related manner, the disclosed methods can be used to Example of methods for transforming microalgae, in addition shield human skin or lip tissue from UV light radiation. The to those demonstrated in the Examples section below, includ UV radiation may comprise UVA and/or UVB. The method ing methods comprising the use of exogenous and/or endog may comprise applying a composition of the disclosed inven enous promoters that are active in microalgae, and antibiotic tion to skin or a skin tissue in an effective or Sufficient amount 30 resistance genes functional in microalgae, have been to shield, at least in part, the skin from UV radiation. In some described. Seeforexample: Curr Microbiol. 1997 December; embodiments, the amount is that which reduces thymidine 35(6):356-62 (Chlorella vulgaris); Mar Biotechnol (NY). dimer formation and/or Sunburn. In an alternative embodi 2002 January; 4(1):63-73 (Chlorella ellipsoidea); Mol Gen ment, a composition of the invention may be applied in an Genet. 1996 Oct. 16:252(5):572-9 (Phaeodactylum tricomu effective or sufficient amount, such as that which reduces 35 tum); Plant Mol. Biol. 1996 April; 31(1):1-12 (Volvox cart further UV-mediated damage, to treat skin that has been dam eri): Proc Natl AcadSci USA. 1994 Nov. 22:91(24): 11562-6 aged by UV radiation. An additional non-limiting example is (Volvox carteri); Falciatore A, Casotti R, Leblanc C, Abrescia a method of for treating skin to reduce the risk of skin cancer C, Bowler C, PMID: 10383998, 1999 May: 1(3):239-251 induced by sunlight or UV radiation. (Laboratory of Molecular Plant Biology, Stazione Zoologica, The polysaccharide compositions may be in the form of a 40 Villa Comunale, 1-801 21 Naples, Italy) (Phaeodactylum tri sterile and/or non-pyrogenic injectable preparation, for comutum and Thalassiosira weissflogii); Plant Physiol. 2002 example, as a sterile injectable aqueous or oleaginous Sus May: 129(1): 7-12. (Porphyridium sp.); Proc Natl Acad Sci pension. This Suspension may be formulated according to USA. 2003 Jan. 21; 100(2):438-42. (Chlamydomonas rein techniques known in the art using Suitable dispersing or wet hardtii); Proc Natl Acad Sci USA. 1990 February: 87(3): ting agents (such as, for example, Tween 80) and Suspending 45 1228-32. (Chlamydomonas reinhardtii); Nucleic Acids Res. agents. The sterile injectable preparation may also be a sterile 1992 Jun. 25; 20(12):2959-65; Mar Biotechnol (NY). 2002 injectable solution or Suspension in a non-toxic parenterally January;4(1):63-73 (Chlorella); Biochem Mol Biol Int. 1995 acceptable diluent or solvent, for example, as a solution in August; 36(5):1025-35 (Chlamydomonas reinhardtii); J. 1,3-butanediol. Among the acceptable vehicles and solvents Microbiol. 2005 August: 43(4):361-5 (Dunaliella); Yi Chuan that may be employed are mannitol, water, Ringers Solution 50 Xue Bao. 2005 April: 32(4):424-33 (Dunaliella); Mar Bio and isotonic sodium chloride solution. In addition, sterile, technol (NY). 1999 May: 1(3):239-251. (Thalassiosira and fixed oils are conventionally employed as a solvent or Sus Phaedactylum); Koksharova, Appl Microbiol Biotechnol pending medium. For this purpose, any bland fixed oil may be 2002 February: 58(2):123-37 (various species); Mol Genet employed including synthetic mono- or diglycerides. Fatty Genomics. 2004 February; 271 (1):50-9 (Thermosynechococ acids, such as oleic acid and its glyceride derivatives are 55 cus elongates); J. Bacteriol. (2000), 182, 211-215: FEMS useful in the preparation of injectables, as are natural phar Microbiol Lett. 2003 Apr. 25: 221(2):155-9: Plant Physiol. maceutically-acceptable oils, such as olive oil or castor oil, 1994 June; 105(2): 635-41; Plant Mol. Biol. 1995 December; especially in their polyoxyethylated versions. These oil solu 29(5):897-907 (Synechococcus PCC 7942); Mar Pollut Bull. tions or Suspensions may also contain a long-chain alcohol 2002:45(1-12):163–7 (Anabaena PCC 7120): Proc Natl Acad diluent or dispersant such as Ph. Helv or a similar alcohol. 60 Sci USA. 1984 March; 81 (5):1561-5 (Anabaena (various Sterile injectable polysaccharide compositions preferably strains)): Proc Natl Acad Sci USA. 2001 Mar. 27: 98(7): contain less than 1% protein as a function of dry weight of the 4243-8 (Synechocystis); Wirth, Mol Gen Genet. 1989 March: composition, more preferably less than 0.1% protein, more 216(1):175-7 (various species); Mol Microbiol, 2002 June; preferably less than 0.01% protein, less than 0.001% protein, 44(6):1517-31 and Plasmid, 1993 September; 30(2):90-105 less than 0.0001% protein, more preferably less than 65 (Fremyella diplosiphon); Hall et al. (1993) Gene 124: 75-81 0.00001% protein, more preferably less than 0.000001% pro (Chlamydomonas reinhardtii); Gruber et al. (1991). Current tein. Micro. 22: 15-20; Jarvis et al. (1991) Current Genet. 19: US 8,932,652 B2 65 66 317-322 (Chlorella); for additional promoters see also Table and 6,110,719). Other examples of second proteins include 1 from U.S. Pat. No. 6,027,900). structural proteins from mammalian skin Such as collagen Suitable promoters may be used to express a nucleic acid and elastin. Assays such as wesytern blot and ELISAS can be sequence in microalgae. In some embodiments, the sequence used to confirm the presence of the second protein in the is that of an exogenous gene or nucleic acid. In some embodi biomass as well as when it is attached to a purified polysac ments the exogenous gene can encode a Superoxide dismu charide. Polysaccharides with fusion proteins bound can be tase (SOD) or an SOD fusion. In cases of an exogenous purified as in Example 2. activity assays for proteins such as nucleic acid coding sequence, the codon usage may be phytases and Superoxide dismutase are well known in the art. optionally optimized in whole or in part to facilitate expres One advantage to a fusion is that the bioactivity of the sion in microalgae. 10 polysaccharide and the bioactivity from the protein can be In some embodiments the invention includes cells of the combined in a product without increasing the manufacturing genus Porphyridium that have been stably transformed with a cost over only purifying the polysaccharide. As a non-limit vector containing a selectable marker gene in operable link ing example, the potent antioxidant properties of a Porphy age with a promoter active in microalgae. In other embodi ridium polysaccharide can be combined with the potent anti ments the invention includes cells of the genus Porphyridium 15 oxidant properties of Superoxide dismutase in a fusion, that have been stably transformed with a vector containing a however the polysaccharide:Superoxide dismutase combina selectable marker gene in operable linkage with a promoter tion can be isolated to a high level of purity using tangential endogenous to a member of the Rhodophyte order. Such flow filtration. In another non-limiting example, the potent promoters include SEQID NOS: 6, 20 and 21, promoters from antiviral properties of a Porphyridium polysaccharide can be the genome of Chondrus crispus (Genbank accession number added to the potent neutralizing activity of recombinant anti Z47547), promoters from the genome of Cyanidioschyzon bodies fused to the protein (SEQ ID NO:15) that tightly merolae (see for example Matsuzaki, M. et al. Nature 428, associates with the polysaccharide. 653-657 (2004); Plant Physiology 137:567-585 (2005): Preferred carbohydrate transporters for expression in Por entire sequence available at http://merolae.biol.s.lu phyridium are SEQID NOs: 33-35 and 38-40. tokyo.ac.jp/db/chromosome.cgi). In other embodiments the 25 In other embodiments, the invention includes genetic invention includes cells of the genus Porphyridium that have expression methods comprising the use of an expression vec been stably transformed with a vector containing a selectable tor. In one method, a microalgal cell. Such as a Porphyridium marker gene in operable linkage with a promoter other than a cell, is transformed with a dual expression vector under con CMV promoter such as that found in PCT application ditions wherein vector mediated gene expression occurs. The WO2006013572. 30 expression vector may comprise a resistance cassette com In other embodiments, the invention provides for the prising a gene encoding a protein that confers resistance to an expression of a protein sequence found to be tightly associ antibiotic such as Zeocin, operably linked to a promoteractive ated with microalgal polysaccharides. One non-limiting in microalgae. The vector may also comprise a second expres example is the protein of SEQ ID NO: 15, which has been sion cassette comprising a second proteinto a promoteractive shown to be tightly associated with, but not covalently bound 35 in microalgae. The two cassettes are physically linked in the to, the polysaccharide from Porphyridium sp. (see J. Phycol. vector. The transformed cells may be optionally selected 40: 568-580 (2004)). When Porphyridium culture media is based upon the ability to grow in the presence of the antibiotic Subjected to tangential flow filtration using a filter containing or other selectable marker under conditions wherein cells a pore size well in excess of the molecular weight of the lacking the resistance cassette would not grow, Such as in the protein of SEQID NO: 15, the polysaccharide in the retentate 40 dark. The resistance cassette, as well as the expression cas contains detectable amounts of the protein, indicating its tight sette, may be taken in whole or in part from another vector association with the polysaccharide. The calculated molecu molecule. lar weight of the protein is approximately 58 kD, however In one non-limiting example, a method of expressing an with glycosylation the protein is approximately 66 kD. exogenous gene in a cell of the genus Porphyridium is pro Such a protein may be expressed directly such that it will be 45 vided. The method may comprise operably linking a gene present with the polysaccharides of the invention or encoding a protein that confers resistance to the antibiotic expressed as part of a fusion or chimeric protein as described Zeocinto a promoteractive in microalgae to form a resistance herein. As a fusion protein, the portion that is tightly associ cassette; operably linking a gene encoding a second protein to ated with a microalgal polysaccharide effectively links the a promoter active in microalgae to form a second expression other portion(s) to the polysaccharide. A fusion protein may 50 cassette, wherein the resistance cassette and second expres comprise a second protein or polypeptide, with a homog sion cassette are physically connected to form a dual expres enous or heterologous sequence. A homogenous sequence sion vector, transforming the cell with the dual expression would result in a dimer or multimer of the protein while a vector; and selecting for the ability to survive in the presence heterologous sequence can introduce a new functionality, of at least 2.5 lug/ml Zeocin, preferably at least 3.0 ug/ml including that of a bioactive protein or polypeptide. 55 Zeocin, and more preferably at least 3.5 ug/ml Zeocin, more Non-limiting examples of the second protein include an preferably at least 5.0 ug/ml Zeocin. enzyme. In optional embodiments, the enzyme is Superoxide In additional aspects, the expression of a protein that pro dismutase, such as that has at least about 60% amino acid duces Small molecules in microalgae is included and identity with the sequence of SEQID NO: 12, SEQID NO: described. Some genes that can be expressed using the meth 13, and proteins from Table 21 as non-limiting examples. In 60 ods provided herein encode enzymes that produce nutraceu Some embodiments, the Superoxide dismutase has at least tical Small molecules such as lutein, Zeaxanthin, and DHA. about 70%, at least about 75%, at least about 80%, at least Preferably the genes encoding the proteins are synthetic and about 85%, at least about 90%, at least about 95%, or at least are created using preferred codons on the microalgae in which about 98%, or higher, amino acid identity with the sequence the gene is to be expressed. For example, enzyme capable of of SEQID NO:12 or 13 or a protein from Table 21. In other 65 turning EPA into DHA are cloned into the microalgae Por embodiments, the enzyme is a phytase (such as GenBank phyridium sp. by recoding genes to adapt to Porphyridium sp. accession number CAB91845 and U.S. Pat. Nos. 6,855,365 preferred codons. For examples of such enzymes see Nat US 8,932,652 B2 67 68 Biotechnol. 2005 August; 23(8): 1013-7. For examples of similarity between two sequences (see, e.g., Karlin & Alts enzymes in the carotenoid pathway see SEQID NOs: 18 and chul, Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One 19 and Table 22. The advantage to expressing Such genes is measure of similarity provided by the BLAST algorithm is that the nutraceutical value of the cells increases without the smallest sum probability (P(N)), which provides an indi increasing the manufacturing cost of producing the cells. cation of the probability by which a match between two For sequence comparison to determine percent nucleotide nucleotide or amino acid sequences would occur by chance. or amino acid identity, typically one sequence acts as a ref For example, a nucleic acid is considered similar to a refer erence sequence, to which test sequences are compared. ence sequence if the Smallest Sum probability in a comparison When using a sequence comparison algorithm, test and ref of the test nucleic acid to the reference nucleic acid is less than erence sequences are input into a computer, Subsequence 10 coordinates are designated, if necessary, and sequence algo about 0.1, more preferably less than about 0.01, and most rithm program parameters are designated. The sequence com preferably less than about 0.001. parison algorithm then calculates the percent sequence iden It should be apparent to one skilled in the art that various tity for the test sequence(s) relative to the reference sequence, embodiments and modifications may be made to the inven based on the designated program parameters. 15 tion disclosed in this application without departing from the Optimal alignment of sequences for comparison can be Scope and spirit of the invention. All publications mentioned conducted, e.g., by the local homology algorithm of Smith & herein are cited for the purpose of describing and disclosing Waterman, Adv. Appl. Math. 2:482 (1981), by the homology reagents, methodologies and concepts that may be used in alignment algorithm of Needleman & Wunsch, J. Mol. Biol. connection with the present invention. Nothing herein is to be 48:443 (1970), by the search for similarity method of Pearson construed as an admission that these references are prior artin & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by relation to the inventions described herein. computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics EXAMPLES Software Package, Genetics Computer Group, 575 Science Dr. Madison, Wis.), or by visual inspection (see generally 25 Example 1 Ausubel et al., Supra). Another example of algorithm that is suitable for determin Growth of Porphyridium cruentum and Porphyridium sp. ing percent sequence identity and sequence similarity is the Porphyridium sp. (strain UTEX 637) and Porphyridium BLAST algorithm, which is described in Altschul et al., J. cruentum (strain UTEX 161) were inoculated into autoclaved Mol. Biol. 215:403-410 (1990). Software for performing 30 2 liter Erlenmeyer flasks containing an artificial seawater BLAST analyses is publicly available through the National media: Center for Biotechnology Information (http://www.ncbi. 1495 ASW medium recipe from the American Type Culture nlm.nih.gov/). This algorithm involves first identifying high Collection scoring sequence pairs (HSPs) by identifying short words of (components are per 1 liter of media) length W in the query sequence, which either match or satisfy 35 some positive-valued threshold score T when aligned with a NaCl 27.0 g word of the same length in a database sequence. T is referred MgSO4·7H2O 6.6 g. to as the neighborhood word score threshold (Altschulet al., MgCl,6HO 5.6 g. supra.). These initial neighborhood word hits act as seeds for CaCl2.H2O 1.5 g. initiating searches to find longer HSPs containing them. The 40 KNO. 1.0 g KH2PO 0.07 g. word hits are then extended in both directions along each NaHCO, 0.04 g sequence for as far as the cumulative alignment score can be 1.OM Tris-HCl buffer, pH 7.6 20.0 ml increased. Cumulative scores are calculated using, for nucle Trace Metal Solution (see below) 1.0 ml Chelated Iron Solution (see below) 1.0 ml otide sequences, the parameters M (reward score for a pair of Distilled water bring to 1.0 L matching residues; always >0) and N (penalty score for mis 45 matching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Trace Metal Solution: Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X 50 ZnCl2 4.0 mg from its maximum achieved value; the cumulative score goes HBO 60.0 mg to zero or below, due to the accumulation of one or more CoCl26H2O 1.5 mg negative-scoring residue alignments; or the end of either CuCl2-2HO 4.0 mg sequence is reached. For identifying whether a nucleic acid or MnCl4H2O 40.0 mg (NH4)Mo,O4H2O 37.0 mg polypeptide is within the scope of the invention, the default Distilled water 100.0 ml parameters of the BLAST programs are suitable. The 55 BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, Chelated Iron Solution: M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLO 60 FeC14HO 240.0 mg SUM62 scoring matrix. The TBLATN program (using pro 0.05M EDTA, pH 7.6 100.0 ml tein sequence for nucleotide sequence) uses as defaults a word length (W) of 3, an expectation (E) of 10, and a BLO Media was autoclaved for at least 15 minutes at 121°C. SUM 62 scoring matrix. (see Henikoff & Henikoff, Proc. Inoculated cultures in 2 liter flasks were maintained at Natl. Acad. Sci. USA 89:10915 (1989)). 65 room temperature on stir plates. Stir bars were placed in the In addition to calculating percent sequence identity, the flasks before autoclaving. A mixture of 5% CO and air was BLAST algorithm also performs a statistical analysis of the bubbled into the flasks. Gas was filter sterilized before entry. US 8,932,652 B2 69 70 The flasks were under 24 hour illumination from above by visual observation and DMMB assay. The time point 0 standard fluorescent lights (approximately 150 uE/m/s). sample represents the Solvent-available polysaccharide con Cells were grown for approximately 12 days, at which point centration in the cell suspension before the cells were sub the cultures contained approximately of 4x10 cells/mL. jected to Sonication. This was the baseline polysaccharide 5 value for the experiments. Example 2 The following Sonication parameters were set: power level=8, 20 seconds ON/20 seconds OFF (Misonix 3000 Dense Porphyridium sp. and Porphyridium cruentum cul Sonicator with flat probe tip). The container with the biomass tures were centrifuged at 4000 ref. The supernatant was sub was placed in an ice bath to prevent overheating and the ice jected to tangential flow filtration in a Millipore Pellicon 2 10 was replenished as necessary. The sample was prepared as device through a 1000 kD regenerated cellulose membrane follows for visual observation and DMMB assay: 100 uL of (filter catalog number P2C01MC01). Approximately 4.1 the biomass sample+900 uL dHO was labeled as dilution 1. liters of Porphyridium cruentum and 15 liters of Porphy 100 LA, of (i) dilution 1+900 uL dHO for cell observation ridium sp. Supernatants were concentrated to a Volume of and DMMB assay. approximately 200 ml in separate experiments. The concen 15 Sonication Experiment 1 trated exopolysaccharide solutions were then diafiltered with In the first experiment the sample was Sonicated for a total 10 liters of 1 mM Tris (pH 7.5). Theretentate was then flushed time period of 60 minutes, in 5-minute increments (20 sec with 1 mM Tris (pH 7.5), and the total recovered polysaccha onds ON/20 seconds OFF). The data is presented in Tables 4, ride was lyophilized to completion. Yield calculations were 5 and 6. The plots of the absorbance results are presented in performed by the dimethylmethylene blue (DMMB) assay. FIG. 4. The lyophilized polysaccharide was resuspended in deion ized water and protein was measured by the bicinchoninic TABLE 4 acid (BCA) method. Total dry product measured after lyo philization was 3.28 g for Porphyridium sp. and 2.0 g for SONICATION RECORD - EXPERIMENT 1. Porphyridium cruentum. Total protein calculated as a per 25 centage of total dry product was 12.6% for Porphyridium sp. Time point and 15.0% for Porphyridium cruentum. Serhi (min) Observations 1 O Healthy red cells Example 3 5 Red color disappeared, Small greenish circular particles 30 3 10 Small particle, Smaller than 5 minute TP Increasing Solvent-available Polysaccharide 4 15 Small particle, Smaller than 10 minute TP. Same A measured mass (approximately 125 grams) of freshly observation as 10 minute time harvested Porphyridium sp. cells, resuspended in a minimum 5 2O Similar to 15 minute TP. Small particles; empty circular shells in the field of vision amount of dHO sufficient to allow the cells to flow as a 6 25 Similar to 20 minute TP liquid, was placed in a container. The cells were subjected to 35 7 30 Similar to 25 minute TP, particles less numerous increasing amounts of Sonication over time at a predeter 8 35 Similar to 30 minute TP 9 40 Similar to 35 minute TP mined Sonication level. Samples were drawn at predeter 10 45 Similar to 40 minute TP mined time intervals, Suspended in measured Volume of 11 50 Very few shells, mostly fine particles dHO and diluted appropriately to allow visual observation 12 55 Similar to 50 minute TP under a microscope and measurement of polysaccharide con 40 13 60 Fine particles, hardly any shells centration of the cell suspension using the DMMB assay. A plot was made of the total amount of time for which the TP = time point, biomass was Sonicated and the polysaccharide concentration of the biomass Suspension. Two experiments were conducted TABLE 5 with different time intervals and total time the sample was 45 Subjected to Sonication. The first data set from Sonication STANDARD CURVE RECORD -- SONICATION EXPERIMENT 1. experiment 1 was obtained by Subjecting the sample to Soni cation for a total time period of 60 minutes in 5 minute Absorbance (AU) Concentration (Ig) increments. The second data set from Sonication experiment O Blank, O 2 was obtained by Subjecting the sample to Sonication for a 50 O.O2 O.25 O.O3 O.S total time period of 6 minutes in 1-minute increments. The O.OS 0.75 data, observations and experimental details are described O.O7 1.O below. Standard curves were generated using TFF-purified, O.09 1.25 lyophilized, weighed, resuspended Porphyridium sp. exopolysaccharide. 55 General Parameters of Sonication Experiments 1 and 2 TABLE 6 Cells were collected and volume of the culture was mea sured. The biomass was separated from the culture solution Record of Sample Absorbance versus Time Points - Sonication by centrifugation. The centrifuge used was a Forma Scientific Experiment 1 60 Centra-GP8R refrigerated centrifuge. The parameters used SAMPLE Solvent-Available for centrifugation were 4200 rpm, 8 minutes, rotori218. Fol TIME POINT Polysaccharide lowing centrifugation, the biomass was washed with dH2O. (MIN) (Ig) The Supernatant from the washings was discarded and the O O.23 pelleted cell biomass was collected for the experiment. 5 1.9S A sample of 100 uL of the biomass Suspension was col 65 10 2.16 lected at time point O (OTP) and suspended in 900 uL dH.O. 15 2.03 The suspension was further diluted ten-fold and used for US 8,932,652 B2 71 72 TABLE 6-continued TABLE 8
Record of Sample Absorbance versus Time Points - Sonication STANDARD CURVE RECORD - SONICATION EXPERIMENT 2 Experiment 1 Absorbance (AU) Concentration (Ig)
SAMPLE Solvent-Available -OOO1 Blank, O O.O17 O.25 TIME POINT Polysaccharide O.O31 O.S (MIN) (Ig) O.049 0.75 O.O645 1.O 2O 186 10 O.O79 1.25 25 1.97 30 1.87 35 2.35 TABLE 9 40 1.47 45 2.12 15 Record of Sample Absorbance versus Time Points - Sonication 50 1.84 Experiment 2 55 2.1 SAMPLE Solvent-Available 60 2.09 TIME POINT Polysaccharide (MIN) (Ig) O O.O63 The plot of polysaccharide concentration versus Sonication 1 O6 time points is displayed above and in FIG. 4. Solvent-avail 2 1.04 3 1.41 able polysaccharide concentration of the biomass (cell) sus 4 1.59 pension reaches a maximum value after 5 minutes of Sonica 5 1.74 tion. Additional Sonication in 5-minute increments did not 25 6 1.78 result in increased solvent-available polysaccharide concen tration. The value of the solvent-available polysaccharide Homogenization by Sonication of the biomass resulted in increases gradually up to the 5 minute time point as shown in an approximately 10-fold increase in solvent-available 30 Table 9 and FIG. 5. polysaccharide concentration of the biomass Suspension, indicating that homogenization significantly enhances the Example 4 amount of polysaccharide available to the solvent. These results demonstrate that physically disrupted compositions of Alcohol Precipitation Porphyridium for oral or other administration provide novel 35 Porphyridium sp. culture was centrifuged at 4000 ref and and unexpected levels or polysaccharide bioavailability com Supernatant was collected. The Supernatant was divided into pared to compositions of intact cells. Visual observation of six 30 ml aliquots. Three aliquots were autoclaved for 15 min the samples also indicates rupture of the cell wall and thus at 121°C. After cooling to room temperature, one aliquot was release of insoluble cell wall-bound polysaccharides from the mixed with methanol (58.3% vol/vol), one was mixed with cells into the Solution that is measured as the increased 40 ethanol (47.5% vol/vol) and one was mixed with isopropanol polysaccharide concentration in the biomass Suspension. (50% vol/vol). The same concentrations of these alcohols Sonication Experiment 2 were added to the three Supernatant aliquots that were not autoclaved. Polysaccharide precipitates from all six samples In the second experiment the sample was Sonicated for a were collected immediately by centrifugation at 4000 refat total time period of 6 minutes in 1-minute increments. The 45 20° C. for 10 min and pellets were washed in 20% of their data is presented in Tables 7, 8 and 9. The plots of the absor respective alcohols. Pellets were then dried by lyophilization bance results are presented in FIG. 5. and resuspended in 15 ml deionized water by placement in a 60° C. water bath. Polysaccharide pellets from non-auto TABLE 7 50 claved samples were partially soluble or insoluble. Polysac SONICATION EXPERIMENT 2 charide pellets from autoclaved ethanol and methanol pre cipitation were partially soluble. The polysaccharide pellet Time point obtained from isopropanol precipitation of the autoclaved Seri (min) Observations Supernatant was completely soluble in water. 55 1 O Healthy red-brown cells appear circular Example 5 2 1 Circular particles scattered in the field of vision with few healthy cells. Red color has mostly disappeared from cell bodies. Monosaccharide Analysis 3 2 Observation similar to time point 2 minute. Approximately 10 milligrams of purified polysaccharide 4 3 Very few healthy cells present. Red color has disappeared and the concentration of particles closer in size to whole 60 from Porphyridium sp. and Porphyridium cruentum (de cells has decreased dramatically. scribed in Example 3) were subjected to monosaccharide 5 4 Whole cells are completely absent. The particles are analysis. Smaller and fewer in number. Monosaccharide analysis was performed by combined gas 6 5 Observation similar to time point 5 minute. 7 6 Whole cells are completely absent. Large particles are chromatography/mass spectrometry (GC/MS) of the per-O- completely absent. 65 trimethylsilyl (TMS) derivatives of the monosaccharide methyl glycosides produced from the sample by acidic methanolysis. US 8,932,652 B2 73 74 Methylglycosides prepared from 500 lug of the dry sample Example 7 provided by the client by methanolysis in 1 M HCl in metha nol at 80°C. (18-22 hours), followed by re-N-acetylation with Generation of Protein-free Polysaccharide pyridine and acetic anhydride in methanol (for detection of Porphyridium sp. was grown as described. 1 liters of cen amino Sugars). The samples were then per-O-trimethylsily 5 trifuged Porphyridium sp. culture Supernatant was autoclaved lated by treatment with Tri-Sil (Pierce) at 80° C. (30 mins). at 121°C. for 15 minutes and then treated with 10% protease These procedures were carried out as previously described (Sigma catalog number P-5147; protease treatment amount described in Merkle and Poppe (1994) Methods Enzymol. relative to protein content of the Supernatant as determined by 230:1-15; York, et al. (1985) Methods Enzymol. 118:3-40. BCA assay). The protease reaction proceeded for 4 days at GC/MS analysis of the TMS methyl glycosides was per 10 37° C. The solution was then subjected to tangential flow formed on an HP 5890 GC interfaced to a 5970 MSD, using filtration in a Millipore Pellicon(R) cassette system using a 0.1 a Supelco DB-1 fused silica capillary column (30m 0.25 mm micrometer regenerated cellulose membrane. The retentate ID). Monosaccharide compositions were determined as fol was diafiltered to completion with deionized water. No pro lows: tein was detected in the diafiltered retentate by the BCA assay. 15 See FIG. 6. TABLE 10 Optionally, the retentate can be autoclaved to achieve ste rility if the filtration system is not sterile. Optionally the Forphyridium sp. monosaccharide analysis sterile retentate can be mixed with pharmaceutically accept able carrier(s) and filled in vials for injection. Glycosyl residue Mass (Ig) Mole % Optionally, the protein free polysaccharide can be frag Arabinose (Ara) n.d. n.d. mented by, for example, Sonication to reduce viscosity for Rhamnose (Rha) 2.7 1.6 parenteral injection as, for example, an antiviral compound. Fucose (Fuc) n.d. n.d. Xylose (Xyl) 70.2 44.2 Preferably the sterile polysaccharide is not fragmented when Glucuronic acid (GlcA) n.d. n.d. prepared for injection as a joint lubricant. Galacturonic acid (GalA) n.d. n.d. 25 Mannose (Man) 3.5 1.8 Example 8 Galactose (Gal) 65.4 34.2 Glucose (Glc) 34.7 18.2 N-acetylgalactosamine (GalNAc) n.d. n.d. Heterotrophic Growth of Porphyridium N-acetylglucosamine (GlcNAc) trace trace Cultures of Porphyridium sp. (UTEX 637) and Porphy 30 ridium cruentum (strain UTEX 161) were grown, to a density X = 176.5 of4x10° cells/mL, as described in Example 1. For each strain, about 2x10 cells/mL cells per well (~500 uL) were trans ferred to 11 wells of a 24 well microtiter plate. These wells TABLE 11 contained ATCC 1495 media supplemented with varying 35 concentration of glycerolas follows: 0%, 0.1%, 0.25%, 0.5%, Porphyridium Cruentum monosaccharide analysis 0.75%, 1%, 2%, 3%. 5%, 7% and 10%. Duplicate microtiter Glycosyl residue Mass (Ig) Mole % plates were shaken (a) under continuous illumination of approximately 2400 lux as measured by a VWR Traceable Arabinose (Ara) n.d. n.d. light meter (cat #21800-014), and (b) in the absence of light. Rhamnose (Rha) n.d. n.d. 40 Fucose (Fuc) n.d. n.d. After 5 days, the effect of increasing concentrations of glyc Xylose (Xyl) 148.8 53.2 erol on the growth rate of these two species of Porphyridium Glucuronic Acid (GlcA) 14.8 4.1 in the light was monitored using a hemocytometer. The results Mannose (Man) n.d. n.d. are given in FIG. 2 and indicate that in light, 0.25 to 0.75 Galactose (Gal) 88.3 26.3 Glucose (Glc) 55.0 16.4 percent glycerol Supports the highest growth rate, with an N-acetylglucosamine (GlcNAc) trace trace 45 apparent optimum concentration of 0.5%. N-acetylneuraminic acid (NANA) n.d. n.d. Cells in the dark were observed after about 3 weeks of X = 292.1 growth. The results are given in FIG. 3 and indicate that in Mole % values are expressed as mole percent of total carbohydrate in the sample. complete darkness, 5.0 to 7.0% glycerol supports the highest n.d. = none detected, growth rate, with an apparent optimum concentration of 50 7.0%. Example 6 Example 9 Cosmeceutical Compositions Protein Measurement 55 Porphyridium sp. (UTEX 637) was grown to a density of Porphyridium sp. was grown as described. 2 liters of cen approximately 4x10° cells/mL, as described in Example 1. trifuged Porphyridium sp. culture Supernatant were auto Approximately 50 grams of wet pelleted, and washed cells claved at 121° C. for 20 minutes and then treated with 50% were completely homogenized using approximately 20 min isopropanol to precipitate polysaccharides. Prior to autoclav utes of Sonication as described. The homogenized biomass ing the 2 liters of Supernatant contained 90.38 mg polysac 60 was mixed with carriers including, water, butylene glycol, charide. The pellet was washed with 20% isopropanol and mineral oil, petrolatum, glycerin, cetyl alcohol, propylene dried by lyophilization. The dried material was resuspended glycol dicaprylate/dicaprate, PEG-40 stearate, C11-13 iso in deionized water. The resuspended polysaccharide Solution paraffin, glyceryl Stearate, tri (PPG-3 myristyl ether) citrate, was dialyzed to completion against deionized waterina Spec emulsifying wax, dimethicone, DMDM hydantoin, meth tra/Por cellulose ester dialysis membrane (25,000 MWCO). 65 ylparaben, carbomer 940, ethylparaben, propylparaben, tita 4.24% of the solid content in the solution was proteins as nium dioxide, disodium EDTA, sodium hydroxide, butylpa measured by the BCA assay. raben, and Xanthan gum. The mixture was then further US 8,932,652 B2 75 76 homogenized to form a composition Suitable for topical Cells expressing an endogenous monosaccharide trans administration. The composition was applied to human skin porter, containing a monosaccharide transporter and capable daily for a period of one week. of importing galactose, are cultured in ATCC 1495 media in the light in the presence of 1.0% galactose for approximately Example 10 12 days. Exopolysaccharide is purified as described in Antibiotic Sensitivity Example 2. Monosaccharide analysis is performed as Approximately 4500 cells (300 ul of 1.5x10 cells per ml) described in Example 5. of Porphyridium sp. and Porphyridium cruentum cultures in Cells expressing an endogenous monosaccharide trans liquid ATCC 1495 ASW media were plated onto ATCC 1495 10 porter, containing a monosaccharide transporter and capable ASW agar plates (1.5% agar). The plates contained varying of importing glucuronic acid, are cultured in ATCC 1495 amounts of Zeocin, Sulfometuron, hygromycin and spectino media in the light in the presence of 1.0% glucuronic acid for mycin. The plates were put under constant artificial fluores approximately 12 days. Exopolysaccharide is purified as cent light of approximately 480 lux. After 14 days, plates described in Example 2. Monosaccharide analysis is per were checked for growth. Results were as follows: 15 formed as described in Example 5. Cells expressing an endogenous monosaccharide trans Zeocin porter, containing a monosaccharide transporter and capable of importing glucose, are cultured in ATCC 1495 media in the Conc. (ug/ml) Growth dark in the presence of 1.0% glucose for approximately 12 O.O ------days. Exopolysaccharide is purified as described in Example 2.5 -- 2. Monosaccharide analysis is performed as described in S.O Example 5. 7.0 Cells expressing an endogenous monosaccharide trans 25 porter, containing a monosaccharide transporter and capable of importing xylose, are cultured in ATCC 1495 media in the Hygromycin dark in the presence of 1.0% xylose for approximately 12 days. Exopolysaccharide is purified as described in Example Conc. (ug/ml) Growth 2. Monosaccharide analysis is performed as described in 30 O.O ------Example 5. S.O ------1O.O ------Cells expressing an endogenous monosaccharide trans SO.O ------porter, containing a monosaccharide transporter and capable of importing galactose, are cultured in ATCC 1495 media in 35 the dark in the presence of 1.0% galactose for approximately 12 days. Exopolysaccharide is purified as described in Specinomycin Example 2. Monosaccharide analysis is performed as described in Example 5. Conc. (ug/ml) Growth Cells expressing an endogenous monosaccharide trans 40 O.O ------porter, containing a monosaccharide transporter and capable 1OOO ------of importing glucuronic acid, are cultured in ATCC 1495 2SO.O ------media in the dark in the presence of 1.0% glucuronic acid for 750.O ------approximately 12 days. Exopolysaccharide is purified as described in Example 2. Monosaccharide analysis is per After the initial results above were obtained, a titration of 45 formed as described in Example 5. Zeocin was performed to more accurately determine growth levels of Porphyridium in the presence of Zeocin. Porphy ridium sp. cells were plated as described above. Results are Example 12 shown in FIG. 8. 50 Increasing Solvent-available Polysaccharide Example 11 128 mg of intact lyophilized Porphyridium sp. cells were ground with a mortar/pestle. The sample placed in the mortar Nutritional Manipulation to Generate Novel Polysaccharides pestle was ground for 5 minutes. 9.0 mg of the sample of the Cells expressing an endogenous monosaccharide trans ground cells was placed in a micro centrifuge tube and Sus porter, containing a monosaccharide transporter and capable 55 of importing glucose, are cultured in ATCC 1495 media in the pended in 1000 uL of dH2O. The sample was vortexed to light in the presence of 1.0% glucose for approximately 12 suspend the cells. 3. days. Exopolysaccharide is purified as described in Example A second sample of 9.0 mg of intact, lyophilized Porphy 2. Monosaccharide analysis is performed as described in ridium sp. cells was placed in a micro centrifuge tube and Example 5. 60 suspended in 1000 uL of dH2O. The sample was vortexed to Cells expressing an endogenous monosaccharide trans Suspend the cells. porter, containing a monosaccharide transporter and capable The suspensions of both cells were diluted 1:10 and of importing xylose, are cultured in ATCC 1495 media in the polysaccharide concentration of the diluted samples was light in the presence of 1.0% xylose for approximately 12 measured by DMMBassay. Upon grinding, the Suspension of days. Exopolysaccharide is purified as described in Example 65 ground cells resulted in an approximately 10-fold increase in 2. Monosaccharide analysis is performed as described in the solvent-accessible polysaccharide as measured by Example 5. DMMB assay over the same quantity of intact cells. US 8,932,652 B2 78 TABLE 12 Example 14 Read 1 Read 2 Avg. Abs Conc. Homogenization of Biomass Sample Description (AU) (AU) (AU) (ig/mL) After Porphyridium biomass grown as described in Blank O -0.004 -0.002 O 5 Example 1 was recovered by centrifugation and washed in 50 ng L Stol., 10 IL: 0.5g O.O3 O.O28 O.O29 NA deionizer water, nitrogen was bubbled through the paste for 100 ng IL. Std., 10 IL; 1.0 Ig O.OS6 0.055 0.0555 NA Whole cell suspension O.O09 O.OO)4 O.OO65 0.0102 30 minutes to displace dissolved oxygen and minimize Sub Ground cell Suspension O.091 O.O72 O.O815 O.128 sequent oxidation. The paste was then passed through a model 110Y Microfluidics Microfluidizer R at 22,000 PSI 10 with cooling, and the process repeated until cell breakage was Reduction in the particle size of the lyophilized biomass by at least 50% as determined by microscopic examination. homogenization in a mortar/pestle results in better Suspen Nitrogen was once again bubbled through the paste, which sion and increase in the solvent-available polysaccharide con was then lyophilized after shell freezing in dry ice ethanol. centration of the cell Suspension. The dried cell mass was then ground to a fine powder with a 15 Braun R) kitchen homogenizer. This process can be performed with decolorized biomass generated as described herein. Example 13 Optionally, preservative(s) and/or carrier(s) suitable for topi cal administration are added to the material, as well as fra Decolorization of Biomass grances and other materials used in the art of skin care product A Porphyridium culture (UTEX 637) was grown as formulation. described in Example 1 except that the culture media con Example 15 tained no Tris and 0.125 g/L potassium nitrate, pH 7.6. The low nitrate culture was grown under approximately 130 uE Hyaluronidase Inhibition m’s' until the color changed from red to yellow-brown, 25 Biotinylated hyaluronic acid (bHA) was covalently which took approximately 3 weeks. After waiting for a further attached to the wells of a 96-well plate. Samples containing three days, the yellow-brown cells were harvested by cen hyaluronidase and various test materials were then added to trifugation, hereinafter referred to as “decolorized biomass” the wells. The hyaluronidase degrades the bound hyaluronic or “decolorized cell pellet'. A deep red cell pellet generated acid, resulting in a decrease in the amount of biotin covalently from cells grown in normal ATCC 1495 ASW media was also 30 linked to the well plate. At the end of the incubation period the generated as described in Example 1. The cell pellets were reaction was stopped and the well plate was washed to remove washed with 0.5 L of distilled water, shell frozen in a dry ice the hyaluronidase. The remaining bA was detected using an acetone bath and lyophilized. avidin bound peroxidase enzyme. When an appropriate Sub Determination of Color strate is added, the peroxidase enzyme generated a color The decolorized cell pellet had a yellow-brown appearance 35 signal in proportion to the amount ofbHA. The color signal (as opposed to cells grown in full ATCC 1495 ASW media was measured spectrophotometrically, and was inversely pro which have a deep red appearance). The lyophilized decol portional to the amount of hyaluronidase activity in the orized cells and lyophilized red cells grown in full ATCC sample. Thus, materials that inhibited hyaluronidase resulted 1495 ASW media were treated identically. 100 mg of lyo in a greater color signal, since more of the bFIA remained philized cell-pellets were resuspended in 4 ml of 1M pH 7.6 40 intact. Also see Frost, G., I., Stem, R. A Microtiter-Based Tris bufferby vigorous Vortexing. The Suspensions were soni Assay for Hyaluronidase Activity Not Requiring Specialized cated on ice using a Misonix 3000 sonicator equipped with a Reagents. Analytical Biochemistry 251, 263-269: 1997. micro-tip probe set at a power level of 6.5 for 90 seconds, Preparation of Test Material Extracts pulsing for 30 seconds on 20 seconds off (3 cycles). The cell Test Material A was supplied as a powder type material. debris was pelleted by centrifugation in a microcentrifuge at 45 For this study, 100 mg of this material was combined with 14,000 rpm for 5 minutes and the supernatant decanted. This either 5 ml of ethanol or 5 ml of ultrapure water in 15 ml procedure was repeated twice more with 1M pH 7.6 Tris centrifuge tubes. After combining, the mixtures were Vor buffer, and finally with 6Murea. No red color was observed in texed, then placed onto a rocking platform for approximately cell pellets or supernatant from the decolorized biomass after 30 minutes at room temperature, and then centrifuged at the second extraction or from the cells grown in full ATCC 50 1,000 RPM for 5 minutes. The supernatants were then used at 1495 ASW media after the fourth extraction. The respective the final concentrations listed in the results section. Test Supernatants were combined and brought to a final Volume of Material B was supplied as a thick, viscous solutions (3%). 75 ml with distilled water. Anti-Hyaluronidase Assay: Immobilization of bHA onto 96-well Plates Absorbance spectra of the Supernatants from the decol 55 A solution of sulfo-NHS (0.184 mg/ml) and bha (0.2 orized pellet and the pellet from cells grown in full ATCC mg/ml) was prepared in distilled water. 50 ul of this solution 1495 ASW media were recorded between 510 and 600 nM was then added to the wells of a 96-well Covalink-NH plate. with a Pharmacia Ultraspec III spectrophotometer and a 1 cm A solution of EDC (0.123 mg/ml) was then prepared in dis path length cuvette. tilled water and 50 ul of this solution was added to each well The extinction coefficient for phycoerythrin is 5.26 ml mg/ 60 (which resulted in a final concentration of 10 ug?well bHA cm at 545 nm (see for example Gantt and Lipschultz, Phyco and 6.15ug/well of EDC). The well plate was then incubated bilisomes of Porphyridium cruentum: Biochemistry, 13, overnight at 4+2°C. or for 2 hours at room temperature. After 2960, 1974). The concentration phycoerythrin was calculated the incubation the plate was washed three times with PBS from the optical density at 545 nm (after subtracting the containing 2 MNaCl and 50 mM MgSO. background due to scatter measured at 600 nm) as 46 mg/g 65 Anti-Hyaluronidase Assay dry-weight in cells grown in ATCC 1495 ASW media and 4.7 Prior to the assay, the well plate was equilibrated with assay mg/g in the decolorized cells. buffer (0.1 M formate pH 4.5, 0.1 M NaCl, 1% Triton US 8,932,652 B2 79 80 X-100, 5 mM saccharolactone). The test materials were pre described in Example 2 from the 120 and 600 mM cultures, to pared in assay buffer at 2x their final concentration (heparin the point where all soluble protein and small molecules were was used as a positive control. 1 mg/ml final concentration). removed. Sulfur content was analyzed according to US EPA After removing the assay buffer from the well plate used for SW846, Method 6010B, Inductively Coupled Plasma equilibration, 50 ul of each of the prepared test materials was 5 Atomic Emission Spectrometry. The polysaccharide purified added to three wells on the well plate, followed by the addi from the 17, 120 and 600 mM cultures contained 3.57, 4.23 tion of 50 ul of assay buffer containing hyaluronidase will be and 5.57% sulfur, respectively. It was observed that polysac added to each well (0.1 mg/ml). Three additional wells were charides with higher percent sulfate by weight exhibited treated with 100 ul of assay buffer alone (without test mate stronger gelling properties than polysaccharides with a lower rials and without hyaluronidase) and served as an index of 10 percent sulfate by weight when the two preparations were Zero hyaluronidase activity. After the addition of the test generated at the same polysaccharide concentration. For materials and enzyme, the plate was incubated for 30 minutes example, at a 1% concentration the polysaccharide contain at room temperature. At the end of the incubation period, 200 ing 5.57% sulfur held its shape and moved as a gelatinous unit ul of 6 M guanidine-HCl was added to each well to terminate 15 whereas the polysaccharide with a 3.57 percent sulfur by the reaction. The plate was then washed three times with PBS weight at 1% flowed as a viscous liquid. The increasedgelling containing 2 MNaCl, 50 mM MgSO and 0.05% Tween 20. properties provide added benefits for skin care compositions During the 30 minute incubation, an avidin/biotin-peroxi as they can form gels in products at lower concentrations. dase complex was prepared in 10.5 ml of PBS containing 0.1% Tween 20 using an ABC kit. This mixture was incubated Example 17 for at least 30 minutes prior to use. After the plate was washed, 100 ul of the avidin/biotin-peroxidase solution was added to Monosaccharide Analysis each well and the plate was incubated for another 30 minutes Porphyridium cruentum was cultured as described in at room temperature. The plate was washed three times with Example 1 except that (a) the amount of KNO per liter of PBS containing 2 MNaCl, 50 mM MgSO and 0.05% Tween 25 media was approximately 150 g/L.; (b) the media contained no 20. After the final wash, 100 ul of substrate solution (one 10 Tris; and (c) the media contained approximately 0.14 g/L mg tablet of OPD in 10 ml of 0.1 Mcitrate-PO, buffer supple KHPO. The cells lost all detectable red coloration after mented with 7.5ul of 30% HO) was added to each well. The approximately three weeks of growth, and turned to a yellow plate was incubated in the dark for 10-20 minutes and then shade. Exopolysaccharide was purified essentially as read at 460 nm using a plate reader. The Substrate solution was 30 described in Example 2. Monosaccharide analysis was per also added to three wells that were not treated with test mate formed essentially as described in Example 5. Monosaccha rials or the avidin/biotin-peroxidase solution and were used ride composition of the exopolysaccharide was as follows: as a blank for the absorbance measurements.
TABLE 13 35 Glycosyl residue Mass (Ig) Mole % Arabinose (Ara) n.d. n.d. Treatment Percent Inhibition Rhamnose (Rha) n.d. n.d. Fucose (Fuc) n.d. n.d. 10%. A Water Extract 86 Xylose (Xyl) 137.8 41.5 5% MATERIALA Water Extract 67 Glucuronic Acid (GlcA) 18.7 4.3 1% MATERIALA Water Extract 29 40 1.5% MATERIALB 93 Mannose (Man) trace trace O.S9 MATERIALB 81 Galactose (Gal) 133.2 33.4 Glucose (Glc) 83.2 20.8 O.1% MATERIALB 70 Unknown Sugar n.d. n.d. 0.1% Heparin 74 N-acetylglucosamine (GlcNAc) n.d. n.d. Negative Control O N-acetylneuraminic acid (NANA) n.d. n.d. 45 Test Material Identification: MATERIAL A: Porphyridium X = 372.9 sp. biomass homogenized (Quadro F10); cells grown as described in Example 1 The exopolysaccharide contained significantly different and Physical Description Red/Purple powder more advantageous monosaccharide composition from those Concentrations Tested: 10%, 5%, 1% (Extracted in either 50 grown under standard conditions shown in Example 5. The ethanol or water) composition is a preferred composition for skin care prod Test Material Identification: 3% MATERIAL B: ucts. For example, the ratio of glucose to Xylose is higher in Exopolysaccahride from Porphyridium sp. purified as the polysaccharide from bleached cells. described in Example 2 Physical Description Light tan, Viscous liquid 55 Example 18 Concentrations Tested: 1.5%, 1%, 0.5%, 0.1% Polysaccharide Bead Production Example 16 Two solutions of polysaccharide from Porphyridium cruentum (0.5% w/v) in water, prepared as described in Sulfated Derivative Polysaccharides 60 Example 2 except flushed with distilled water rather than 1 Porphyridium cruentum and Porphyridium sp. were grown mM Tris, were dried under air flow at 60° C. until converted in artificial seawater media essentially as described in to a solid translucent film. One sample was isolated from Example 1 except that the amount of MgSO was varied. Porphyridium cruentum grown in ATCC 1495 media, while Porphyridium sp. cells were grown in 17 mM MgSO Por the other was from Porphyridium cruentum grown in ATCC phyridium cruentum was grown in 120 mM, 600 mM, 750 65 1495 media with the exception that the KNO was approxi mM, 1M, and 2MMgSO. Cell division occurred at all con mately 0.15 g/L (labeled as “1495 low N”). The resulting centrations. Polysaccharide was purified essentially as films were then heated under vacuum (>25 in Hg) at approxi US 8,932,652 B2 81 82 mately 150° C. overnight to form dried polysaccharide. The pBlusecript KpnI site. The CMV 3'UTR was also in operable dried polysaccharide was then ground in a pestle and mortar. linkage with the ble cDNA. The plasmid was linearized by A third polysaccharide sample (0.5% w/v) in water, isolated KpnI, which does cut in the promoter, ble cDNA, or 3'UTR, from Porphyridium cruentum grown in ATCC 1495 media, prior to transformation. prepared as described in Example 2 except flushed with dis The biolistic particle delivery system PDS 1000/He (Bio tilled water rather than 1 mM Tris, was lyophilized and not Rad, USA) was used for transformation. Porphyridium sp. ground. culture was grown to logarithmic phase (-2x10 cells/mL) in 100 mg of dried, ground polysaccharide from each sample liquid ATCC 1495 media under continuous light (approxi was resuspended in 2.5 ml of water. 100 mg of lyophilized mately 75 umol/photons/m). Cells from this culture were polysaccharide was also resuspended in 2.5 ml of water. The 10 harvested at 4,000 rpm at room temperature. The cell pellet Suspensions were centrifuged at 4,400 rpm in a Form a Sci was washed twice with sterile distilled water. Cells were entific Centra-GP8R refrigerated centrifuge. The parameters resuspended in fresh ATCC 1495 media to the same cell used for centrifugation were 4200 rpm, rotoril 218, 20 minute density i.e. ~2x10 cells/mL and incubated in the dark for 16 spin. As shown in FIG.16(c), there was a swollen, insoluble hours. The dark adapted cells were then harvested at 4000 gel layer and a clear Supernatant in the samples that were 15 rpm at room temperature, resuspended in fresh ATCC 1495 dried at 150° C. but not in the samples that were lyophilized media to a density of -2x10 cells/mL. Approximately 1x10 ordried at 105°C. The polysaccharide dried at 150° C. did not cells were transferred to each ATCC 1495 agarose plate. Filter go into Solution, but rather stayed insoluble despite signifi sterilized DNA from the plasmids was coated onto 550 nm cant Swelling in size. gold particles (catalog number SO4e, Seashell Technology, The percentage insoluble polysaccharide in the 150° C. USA) according to the manufacturer's protocol. For each of dried samples was measured by separating the insoluble and the particle bombardments, 1 ug of plasmid DNA was used. Supernatant fractions, lyophilizing the separated fractions, The negative controls were bombarded in identical fashion and weighing the dried residual polysaccharide from each with gold particles coated with a plasmid containing the Por fraction. The percentage insoluble polysaccharide was then phyridium glycoprotein promoter, SEQ ID NO:21, and the calculated as a percentage of the total polysaccharide from 25 CMV 3'UTR (SEQ ID NO:32), with no zeocin resistance both fractions. While the polysaccharide from the samples gene. Each of the particle bombardments were performed that were originally dried by lyophilization and drying at 105° using 1350 psi rupture disks, at bombardment distance of 9 C. were 100% soluble, the low N and ATCC 1495 polysac cm, and under 28 in. Hg vacuum. The bombarded cells were charide samples dried at 150° C. were 75% and 86% scraped off the plates, and transferred to 100 ml of fresh insoluble, respectively. Independent experiments demon 30 ATCC 1495 media, and shaken under continuous light (ap strated that material dried at 125°C. was completely soluble. proximately 75umol/m) for 3 days. Following recovery, the cells were harvested at 4,000 rpm at room temperature, and Example 19 plated onto ATCC 1495 plates supplemented with 30 ug/mL Zeocin (Invitrogen, Carlsbad, Calif., USA) at a cell density of Polysaccharide Bead Properties 35 1x10" cells/plate. These plates were incubated under light Insoluble polysaccharide preparations, prepared essen (approximately 25umol/m) for 4-5 weeks. Zeocin resistant tially as described in Example 18, were tested for (a) the colonies growing on these plates were scored as transfor ability to swell in size over time and (b) the ability to bind mants and transferred onto fresh ATCC 1495 plates supple soluble polysaccharide and remove it from Solution. mented with 30 ug/mL Zeocin (Invitrogen, Carlsbad, Calif., Samples were resuspended in distilled water as described 40 USA) for growth and analysis. in Example 18. Samples were centrifuged as described in Zeocin resistant colonies appeared after 2-3 weeks. Geno Example 18 at various time points. The volume of the typing with primers specific to the Zeocin resistance gene was insoluble gel layer was measured, followed by resuspension performed on genomic DNA isolated from Zeocin resistant of the material and incubation at room temperature until the colonies. Results from genotyping of one strain (referred to next centrifugation point. Results are shown in FIG. 17. The 45 herein and labeled as “transformant #2 in FIG. 14) indicated results demonstrate that the dried polysaccharide beads con that the Zeocin resistance gene was present. A band of the tinue to Swell in Volume for at least 4 hours and as long as 18 correct size was amplified. Results are shown in FIG. 14 and hours after initial exposure to water. discussed in more detail in Example 20. The concentration of polysaccharide in Solution in the Supernatant was measured at each time point using the 50 Example 21 DMMB assay as described in Example 3. Results are shown in FIG. 17(b). The results demonstrate that the polysaccha Transformation of Porphyridium, Genotyping, and South ride beads bind soluble polysaccharide and remove it from ern Blot Analysis solution for at least 4 hours after initial exposure to water. The The Zeocin resistance plasmid described in Example 20 Swelling and binding of Soluble polysaccharide is a useful 55 and a second plasmid that was identical with the exception property for topical application to human skin and is stimu that it contained a cDNA for a human GLUT1 glucose trans lated by transepidermal water loss. porter (SEQID NO:25) instead of the ble cDNA were com bined in a co-transformation experiment carried out essen Example 20 tially as described in Example 20 except that both the Zeocin 60 resistance and GLUT1 plasmids were both adhered to the Transformation of Porphyridium and Genotyping gold beads. A Zeocin resistant colony (referred to herein as The Porphyridium glycoprotein promoter, SEQID NO:21, transformantil 1) was selected for further analysis. Genomic was cloned in operable linkage with a Zeocin resistance ble DNA was extracted from wild type Porphyridium sp. and cDNA with small amounts of flanking sequence (SEQ ID transformantil 1. NO:37), with the far 5' region of the glycoprotein promoter 65 Genotyping was performed on genomic DNA extracted directly adjacent to the p3lusecript SacI site and the far 3' from wild type, transformantil 1, and transformantil 2 DNA region of the CMV 3'UTR (SEQID NO:32) adjacent to the with plasmid DNA used as a template positive control and US 8,932,652 B2 83 84 water in place of DNA as a template negative control. A rials BM, LS PS, and HSPS were generated and tested as segment of the Porphyridium glycoprotein (GLP) gene pro referenced in this and following Examples. BM is Porphy moter was used as a target positive control. The following ridium sp. biomass, grown essentially as described in primer sets were used for the genotyping PCR: Ble-FWD Example 1, which was washed once with distilled water, run (SEQ ID NO: 26) and B1e-REV (SEQ ID NO: 27), GLP twice through a microfluidizer (Microfluidics Inc., Newton, FWD (SEQID NO:28) and (SEQID NO: 29), GLUT1-FWD Mass., U.S.A.), and lyophilized as follows: using a Microf (SEQ ID NO:30) and GLUT1-REV (SEQID NO: 31). The luidics Microfluidizer R. (model #110Y) pressurized with PCR profile used was as follows: 94° C. denaturation for 5 nitrogen, washed biomass material was pumped through an min: 35 cycles of 94° C. for 30 sec, 51° C. or 60° C. (51° C. 87um orifice at 22,000 psi twice. The product was kept on ice for glycoprotein gene & GLUT1 and 60° C. for ble) for 30 10 at all times. Nitrogen gas was bubbled through the final prod sec, 72°C. for 2 min; 72°C. for 5 min. Results are shown in uct while mixing for 10 min. Snap freeze for storage or shell FIG. 14. FIG. 14(a) demonstrates that the ble gene was freeze and lyophilize. The BM material was then treated as present in both transformants, as the expected 300 bp product described in each example. was generated. FIG. 14(b) demonstrates that the genomic LSPS was polysaccharide from Porphyridium sp. HSPS DNA extraction and amplification was working, as the 15 was polysaccharide from Porphyridium cruentum. Both were expected 948bp glycoprotein promoter fragment was gener purified essentially as described in Example 2. The cells ated. FIG. 14(c) demonstrates that the GLUT1 gene was grown for preparation of LS PS and HS PS were grown present transformant #1, as the expected 325bp product was essentially as described in Example 1 except that the sulfate in generated. DNA ladder was from BioNexus, Inc., All Purpose the media was 17 mM for the LS PS and 600 mM for the HS Hi-Lo DNA Marker, Catalog No: BN2050. PS (also described in Example 16). Specific bands can be amplified from residual plasmid DNA adhered to the outside of cells on transformation plates. Example 23 Additionally, plasmids that have not been linearized can be maintained as episomes for a period of time before being UVB Protective Properties of Materials from Microalgae degraded and can serve as template during PCR despite not 25 TT dimer-UV exposure assay: The testing system used for having been integrated into a chromosome of a host organism. this assay was the Mattek EpiDerm R), a skin model that In both cases, microalgal strains may genotype positive consists of normal human-derived epidermal keratinocytes despite the absence of stable chromosomal integration of the cultured to form a multilayered, highly differentiated model vector. Antibiotic resistant strains are known to arise due to of the human epidermis. For this study, the tissues were mutagenesis caused by chromosomal damage from biolistic 30 treated topically overnight with either test materials, 1 mM particles, electroporation conditions, and random genetic Trolox (positive control), or left untreated (negative control). variation that is known to occur in microbial organisms. On the following day, the tissues were exposed to UVB (300 Southern blot analysis was performed to conclusively con mJ/cm). Following the exposures the DNA was extracted firm the integration of the GLUT1 construct into the genome from the EpiDerm tissues and assayed for thymine dimer of transformant #1. 35 content. Samples of the DNA were immobilized on a solid Southern blot analysis was performed on transformant #1. membrane Support and incubated with an antibody that rec 20 uggenomic DNA from wildtype and transformantil 1 were ognizes thymidine dimers in double stranded DNA. The pri individually digested with Hine II, Sac I, XhoI and separated mary antibody was detected using a secondary antibody con on a 1% agarose gel. DNA was transferred onto Nylon mem jugated to an alkaline phosphatase enzyme followed by the brane (Hybond N-- Amersham Biosciences). A 1495bp frag 40 addition of a Substrate that the alkaline phosphatase enzyme ment containing the entire coding region of the GLUT1 gene uses to generate a chemiluminescent signal. The light gener was used as a probe. DIG labeled probes were generated for ated by this reaction was captured using film with the inten each probe fragment using the DIG High Prime DNA labeling sity of the light signal being proportional to the amount of the and detection Kit according to the manufacturers instructions thymine dimers present in the sample. (Roche). The hybridizing bands were detected using the colo 45 Test material BM was Porphyridium sp. biomass that had rimetric detection reagents provided by the manufacturer. been homogenized with a Microfluidizer(R) twice and then FIG. 15 demonstrates that the GLUT1 plasmid was stably lyophilized. For this study, 100 mg of this material was com integrated into the genome of transformantil 1 while no bined with 5 ml of ultrapure water in 15 ml centrifuge tubes. detectable signal arose from wild type genomic DNA. As After combining, the mixture was Vortexed, then placed onto would be expected for a plasmid integrating into a chromo 50 a rocking platform for approximately 30 minutes at room Some of an organism, the specific band was in a different temperature, and then centrifuged at 1,000 RPM for 5 min position for each different restriction enzyme used to digest utes. The Supernatant was then used at a final concentration of the genomic DNA. This data conclusively demonstrates a 10% and 5%. The two remaining test materials, LSPS and HS species of the genus Porphyridium containing an exogenous PS were supplied as thick, viscous solutions (3 g/100 mL). LS gene encoding a carbohydrate transporter integrated into a 55 PS53 was tested at the final concentrations of 1.5%, 0.5%, and chromosome of the organism. In this embodiment the carbo 0.1%, while PS133 was tested at the final concentration of hydrate transporter gene is in operable linkage with a pro O.1%. moter endogenous to a species of the genus Porphyridium. In Prior to use, the Mattek EpiDerm R) tissues were removed some other embodiments embodiment the carbohydrate from the agarose-shipping tray and placed into a 6-well plate transporter gene is in operable linkage with a promoter 60 containing 0.9 ml of culture medium (Mattek EPI-100 cul endogenous to a Rhodophyte. ture media) according to the manufacturer. The tissues were allowed to incubate for at least 1 hour at 37+2° C. and 5+1% Example 22 CO. After this initial incubation, the culture media was replaced with fresh, pre-warmed EPI-100 media and 100 ul of Production of Materials from Microalgae 65 test material, 1 mM Trolox or PBS (negative control) was Materials from Porphyridium were generated to assess applied. The tissues were then incubated overnight at 37+2 their utility as components of skin care compositions. Mate C. and 5+1% CO. On the following day, the tissues were US 8,932,652 B2 85 86 exposed to 300 m.J/cm of UVB energy at 300 nm. After the respective fluorescence intensity (measured in RFUs or rela UVB exposure the tissues were collected and DNA was tive fluorescence units). A regression analysis was performed immediately extracted. to establish the line that best fits these data points. The RFUs DNA extraction was performed using the Qiagen, DNEasy for each unknown sample were used to estimate the amount Kit. Single tissues were placed into 1.5 ml centrifuge tubes DNA. Mean densitometric values, expressed in optical den containing 180 ul of Lysis Buffer One. After mincing the sity units, were determined for each treatment. tissues with a pair of fine tipped scissors, 20 ul of Proteinase The results for the thymidine dimer assay are presented in K was added to the tube and the tube was incubated overnight Table 14 and FIG.11(a). The values are expressed as optical at 55+2°C. with occasional mixing/vortexing. After the Pro density units. The values are presented as mean teinase K digestion, 200 ul of Lysis Buffer Two was added to 10 valueststandard deviation. the tube and the tube was incubated at 70+2°C. for approxi mately 10 minutes. Next, the DNA was precipitated by the addition of 200 ul of 100% ethanol. The precipitated DNA TABLE 1.4 was washed to remove cellular debris by applying the mixture Thymidine Diner ASSay to a DNEasy Spin Column and centrifuging the sample in a 2 15 ml collection tube at 8,000 RPM for 1 minute. The flow Treatment Optical Density through and collection tube was discarded, and 500 ul of 10% BM 85 - 11 Wash Buffer One was added to the spin column and the 5% BM 140 15 column was placed into a new collection tube and centrifuged 1.5% LSPS 1403 O.S9/o LSPS 152- 16 at 8,000 RPM for 1 minute. The flow through and collection O.1% LSPS 150 13 tube was again discarded, and 500 ul of Wash Buffer Two was O.1% HSPS 16O2 added to the spin column and the column was placed into a 1 mM Trolox 107 - 1 new collection tube and centrifuged at 14,000 RPM for 3 Untreated 1966 minutes. The spin column was then placed into a new 1.5 ml Non-UVB Exposed 394 centrifuge tube and 1100 of Elution Buffer was added to the 25 column. The column was incubated for 1 minute at room The results of this study indicate that all three of the test temperature and then centrifuged at 8,000 RPM for 1 minute. materials significantly reduced the amount of TT dimer for Extracted DNA was quantified via a fluorometric assay. A mation. 10 ul aliquot of the DNA sample was mixed with 1.0 ml of Assay Buffer (2 MNaCl, 50 mM sodium phosphate, pH 7.4) 30 Example 24 and 100 ul of this diluted sample was transferred to a well in ablack 96-well plate. A series of DNA standards (0, 100, 200, Procollagen Synthesis Stimulating Properties of Materials 300,400 and 500 ng) was also transferred to wells in a 96-well from Microalgae plate (in duplicate). Finally, 100 ul of dilute Hoechst 33258 A fibroblast cell culture model was used to assess the dye (0.006 mg/ml in Assay Buffer) was added to each well 35 ability of the polysaccharide from Porphyridium to exert an and the fluorescence intensity of each well was determined effect on procollagen synthesis. Fibroblasts are the main using an excitation wavelength of 355 nm and an emission Source of the extracellular matrix peptides, including the wavelength of 485 nm. structural protein collagen. Procollagen is a large peptide Aliquots of DNA (400 ng in 2xSSC 20x stock SSC: 3 M synthesized by fibroblasts in the dermal layer of the skin and NaCl, 0.3 M sodium citrate, pH 7.0) was loaded onto a 40 is the precursor for collagen. As the peptide is processed to membrane via microfiltration blotting. After loading, the form a mature collagen protein, the propeptide portion is membrane was washed once in 2xSSC and thenbaked for 30 cleaved off (type I C-peptide). Both the mature collagen minutes at 80°C. to crosslink the DNA to the membrane. The protein and the type I C-peptide fragment are then released membrane was then incubated for 1 hour in blocking solution into the extracellular environment. As collagen is synthesized (TBS 20 mM Tris, pH 7.5, 500 mM. NaCl) supplemented 45 the type I C-peptide fragment accumulates into the tissue with 5% non-fat milk protein, 0.2% polyvinylpyrolidone, and culture medium. Since there is a 1:1 stoichiometric ratio 0.2% ficol), and then briefly washed twice in TBS-T (TBS between the two parts of the procollagen peptide, assaying for with 0.1% non-fat milk protein and 0.1% Tween 20). The type I C-peptide reflects the amount of collagen synthesized. membrane was then incubated overnight (4°C.) with an anti Type 1 C-peptide was assayed via an ELISA based method. body that recognizes thymine dimers diluted in TBS-T. On 50 Test material LSPS was supplied as a thick, viscous solution the following day, the membrane was washed 3 times with (3 g/100 mL). This material was used at a 0.1% final concen TBS-T (20 minutes per wash) and then incubated with an tration. The anti-collagen antibody used was Procollagen AP-conjugated secondary antibody for 1-2 hours at room Type I: N-18 antibody, catalog number sc-8785, Santa Cruz temperature. After this incubation period the membrane was Biotechnology. The secondary antibody used was anti-goat washed as described above. Near the end of the final wash the 55 conjugated with alkaline phosphatase catalog number chemiluminescence reagent was prepared. At the end of the sc-2355, Santa Cruz, Biotechnology. last wash, all of the TBS-T was drained from the membrane Fibroblasts were seeded into the individual wells of a 12 and the chemiluminescent Substrate was applied to the mem well plate in 1.0 ml of Fibroblast Growth Media (FGM: brane and it was allowed to sit for approximately 1 minute. DMEM supplemented with 2% FBS, 5 ng/ml human recom The membrane was then wrapped in Saran foil and taped 60 binant growth factor, 5 ug/ml insulin, 50 ug/ml gentamicin, inside of a film cassette. In a dark room a piece of film was and 0.5 ug/ml Amphotericin-B) and incubated overnight at inserted into the cassette and exposed for various amounts of 37+2°C. and 5+1% CO. On the following day the media was tune, starting with 10 seconds and increasing or decreasing in removed via aspiration to eliminate any non-adherent cells appropriate increments, until obtaining the necessary expo and replaced with 1.0 ml of fresh FGM. The cells were grown Sures. After exposure, the films were analyzed via densitom 65 until confluent with a media change every 48 to 72 hours. etry. To quantify the amount of DNA present, a standard curve Upon reaching confluency the cells were treated for 24 hours was generated using known concentrations of DNA and their with DMEM supplemented with 1.5% FBS to wash out any US 8,932,652 B2 87 88 effects from the growth factors included in the normal culture at 1,000 RPM for 5 minutes. The supernatant for each mixture media. After this 24-hour wash out period the cells were was then used at final concentrations indicated in FIG. 12(a). treated with 0.1% LSPS dissolved in FGM with 1.5% FBS. 4 LSPS and HSPS was supplied as a thick, viscous solution (3 mM Sodium butyrate was used as a positive control. g/100 mL). This material was used at final concentrations Untreated cells (negative controls) received FGM with 1.5% 5 indicated in FIG. 12(a). FBS. The cells were incubated for 48 hours and at the end of Fibroblasts were seeded into the individual wells of a 12 the incubation period cell culture medium was collected and well plate in 1.0 ml of Fibroblast Growth Media (FGM) and either stored frozen (-75°C.) or assayed immediately. Mate incubated overnight at 37+2° C. and 5+1% CO. On the rials were tested in triplicate. 10 following day the media was removed via aspiration to elimi A series of type I C-peptide standards was prepared rang nate any non-adherent cells and replaced with 1.0 ml of fresh ing from 0 ng/ml to 640 ng/ml. Next, an ELISA microplate FGM. The cells were grown until confluent, with a media was prepared by removing any unneeded strips from the plate change every 48 to 72 hours. Upon reaching confluency the frame followed by the addition of 100 ul of peroxidase cells were treated for 24 hours with DMEM supplemented labeled anti procollagen type I-C peptide antibody to each 15 with 1.5% FBS to wash out any effects from the growth well used in the assay. Twenty (20) ul of either sample (col factors included in the normal culture media. After this lected tissue culture media) or standard was then added to 24-hour wash out period the cells were treated with the test appropriate wells and the microplate was covered and materials at the specified concentrations dissolved in FGM allowed to incubate for 30.25 hours at 37° C. After the with 1.5% FBS. 4 mM sodium butyrate was used as a positive incubation the wells were aspirated and washed three times control for elastin synthesis. Untreated cells (negative con with 400 ul of wash buffer. After the last wash was removed trols) received FGM with 1.5% FBS. The cells were incu 100 ul of peroxidase substrate solution (hydrogen peroxide+ bated for 48 hours and at the end of the incubation period cell tetramethylbenzidine as a chromagen) was added to each well culture medium was collected and either stored frozen (-75° and the plate was incubated for 15+5 minutes at room tem C.) or assayed immediately. Materials were tested in tripli perature. After the incubation 100 ul of stop solution (1 N 25 Cate. sulfuric acid) was added to each well and the plate was read Soluble C-elastin was dissolved in 0.1 M sodium carbonate using a microplate reader at 450 nm. (pH 9.0) at a concentration of 1.25 g/ml. 150% of this To quantify the amount of procollagen present, a standard solution was then applied to the wells of a 96-well maxisorp curve was generated using known concentrations of procol 30 Nunc plate and the plate was incubated overnight at 4°C. On lagen. A regression analysis was be performed to establish the the following day the wells were saturated with PBS contain line that best fit the data points. Absorbance values for the test ing 0.25% BSA and 0.05% Tween 20. The plate was then and positive control samples was used to determine the incubated with this blocking solution for 1 hour at 37°C. and amount of procollagen present. The values are presented in thenwashed two times with PBS containing 0.05% Tween 20. mean ng/ml+the standard deviation of the mean. 0.1% LSPS 35 increased procollagen synthesis compared to untreated cells, A set of C-elastin standards was generated ranging from 0 as seen below in Table 15 and in FIG.11(b). to 100 ng/ml. 180 ul of either standard or sample was then transferred to a 650 ul microcentrifuge tube. The anti-elastin TABLE 1.5 antibody was the C-21 antibody, catalog number sc-17581, 40 from Santa Cruz, Biotechnology. The secondary antibody Procollagen Assay used was anti-goat conjugated with alkaline phosphatase Treatment Type IC-Peptide ng/ml catalog number sc-2355, Santa Cruz, Biotechnology. An anti elastin antibody solution was prepared (the antibody was O.1% LSPS 1448 - 113 4 mM Na Butyrate 1425 - 81 diluted 1:100 in PBS containing 0.25% BSA and 0.05% Untreated 1151.96 45 Tween 20) and 20 ul of the solution was added to the tube. The tubes were then incubated overnight at 4+2°C. On the fol lowing day, 150 ul was transferred from each tube to the 96-well elastin ELISA plate, and the plate was incubated for Example 24 1 hour at room temperature. The plate was then washed 3 50 times with PBS containing 0.05% Tween 20. After washing, Elastin Synthesis Stimulating Properties of Materials from 200 ul of a solution containing a peroxidase linked secondary Microalgae antibody diluted in PBS containing 0.25% BSA and 0.05% A fibroblast cell culture model was used to assess elastin Tween 20 was added, and the plate was incubated for 1 hour synthesis. Elastin is the main component of a network of at room temperature. After washing the plate three times as elastic fibers that give tissues their ability to recoil after a described above, 200 ul of a substrate solution was added and transient stretch. This protein is released by fibroblasts the plate was incubated for 10 to 30 minutes in the dark at (soluble elastin) into the extracellular space where it is then room temperature. After this final incubation the plate was cross-linked to other elastin proteins to form an extensive read at 460 nm using a plate reader. network of fibers and sheets (insoluble elastin). Soluble elas 60 To quantify the amount of elastin present, a standard curve tin can be readily measured from cell culture medium via a was generated using known concentrations of elastin. A competitive ELISA based method. regression analysis was performed to establish the line that Test material BM was supplied as a powder type material. best fit these data points. Absorbance values for the test and 100 mg of BM was combined with 5 ml of ultrapure water in control samples was used to determine the amount of elastin 15 ml centrifuge tubes. After combining, the mixtures were 65 present in each sample. Both the 0.5% and 0.1% concentra Vortexed, then placed onto a rocking platform for approxi tions of LS PS and HS PS induced an increase in elastin mately 30 minutes at room temperature, and then centrifuged synthesis. US 8,932,652 B2 89 90 Example 25 membrane was wiped clean with a tissue to remove any non-migrating cells and the bottom wells of the chemotaxis Antiinflammatory Properties of Materials from Microal chamber were read using a fluorescent plate reader (485 nm. gae excitation, 530 nm emission). Microalgal polysaccharide materials were tested for their 5 The mean fluorescence of the three wells filled directly ability to influence the migration of polymorphonucellar with fluorescent PMN cells was determined and used to rep (PMN) leukocytes in response to chemotractant substances. resent 100% migration. The mean fluorescence of the test Leukocyte migration is essential for leukocyte accumulation material treated PMN cells were then determined (Test Mate at sites of inflammation. During the inflammatory response, rial Migration). The fluorescent values were corrected for leukocytes migrate towards the source of a chemotractant 10 non-chemotaxis migration by Subtracting the mean fluores Substance in a process called chemotaxis. In vitro methods to cent measurements for the wells where FMLP was not present study chemotaxis often use a membrane based approach, as a chemotractant. Percent PMN migration was then calcu where a chemoattractant is placed on one side of a membrane lated using the following equation: ((Test Material Migration and PMN leukocytes are placed on the other. Chemotaxis is (mean RFU))/(100% Migration (mean RFU)))x100 then measured by quantifying the number of leukocytes 15 The results for the PMN migration assay are presented in which then migrate through the filter towards the chemotrac Table 16 and FIG. 12(b). The values are expressed as mean tant Substance. percent of maximal (100%) migrating cells:tstandard devia For this study, human PMN leukocytes were isolated via tion. The LSPS and HSPS samples reduced PMN migration density centrifugation from freshly drawn blood. The PMN at all concentrations tested. cells were loaded with a Calcein AM, a fluorescent dye. While the PMN cells were being labeled, the bottom chamber of a TABLE 16 chemotaxis chamber was filled with PBS containing FMLP, a chemotractant Substance. A membrane filter was placed over PMN Migration the bottom wells, after which a small aliquot of fluorescently Treatment Percent of Maximal Migration labeled PMN cells was placed on top, along with the LS PS 25 and HSPS test materials and positive control materials. The 1.5% LSPS 7.3 1.O chemotaxis chamber was be incubated and at the end of the O.S9/o LSPS 7.7 O.7 O.1% LSPS 11.10.6 incubation period the fluorescence of the bottom wells was 1.5% HSPS 6.O-O.8 read and used as an index of PMN migration. Test material LS O.S90 HSPS 9.00.4 PS and HSPS, were supplied as thick, viscous solutions (3 30 O.1% HSPS 10.6 - 1.3 g/100 mL). These materials were used at a final concentration 2 ugml Amphotericin B 94 - 0.6 of 1.5%, 0.5% and 0.1%. Untreated 13.50.9 Heparinized whole blood (20-30 ml) was collected from a healthy human donor, layered over a density gradient (Histo paque 1077 and Histopaque 1119) and centrifuged at 400 g 35 Example 26 for 30 min. The PMN rich fraction was removed, washed twice in with phosphate buffered saline (PBS) and then resus Antiinflammatory Properties of Materials from Microal pended in 5.0 ml RPMI-1640 without phenol red supple gae mented with 10% heat-treated fetal calf serum (RPMI-FCS). The pro-inflammatory cytokine interferon-gamma, which Calcein AM (5ug/ml final) was added to the cell suspension 40 is released by activated T-lymphocytes, plays a role in human and then the cells were incubated for 30 min at 37° C. After inflammatory responses. Interferon-gamma also stimulates the incubation, the PMN cells were washed twice with PBS, the enzyme indoleamine-2,3-dioxygenase, which degrades counted and resuspended in RPMI-FCS at 1-5x10 cells/ml. tryptophan to kynurenine. In concert with other pro-inflam A disposable 96-well chemotaxis chamber was used to matory cytokines, interferon-gamma is the most important measure PMN migration (chemotaxis chambers manufac 45 trigger for the formation and release of reactive oxygen spe tured by Neuroprobe Inc., Gaithersburg, Md., catalog number cies (ROS). Interleukin-1 alpha (IL1-C.) is a cytokine that also 101-5). To set up the 96-well chamber, the wells in the micro plays a major role in inflammatory responses. Microalgal plate (bottom chamber) were filled with 29 ul of FMLP (0.1 materials were tested for their ability to suppress secretion of uM) diluted in PBS with 0.1% serum albumin. Negative gamma interferon and IL1-C by human peripheral blood control wells were prepared with PBS and albumin (no FMLP 50 mononuclear cells. added). Amphotericin B (2 ug/ml) was used as a positive Heparinized whole blood (20-30 ml) was collected from a control (inhibits PMN migration). In addition, 3 wells were healthy human donor, layered over a density gradient (Histo filled with 12.5 ul of fluorescently labeled PMN cells and paque 1077 and Histopaque 1119) and centrifuged at 400 g 12.50 of PBS with 0.1% albumin. These latter wells were for 30 min. The PBMC rich fraction was removed and washed used as an index to represent 100% migration by the PMN 55 twice in with 5.0 ml RPMI-1640 without phenol red supple cells. mented with 5% heat-treated fetal calf serum (RPMI-FCS). Test materials were prepared in RPMI-FCS at 2x their After washing, the cells were resuspended in RPMI-FCS at a desired final concentration. RPMI-FCS without test materials final density of 1x10 cells/ml. was used as another negative control. Aliquots of the test Fifty microliters of PBMC cells were added to wells in a materials were combined with an equal volume of the fluo 60 96-well plate (each treatment was tested in triplicate). Next, rescently labeled PMN cells. A chemotaxis membrane was 50 ul of RPMI-FCS supplemented with 2 ug/ml phytohemag then placed over the bottom wells of the chemotaxis chamber glutinin (PHA) and the respective test materials was added to and anchored into place. 25 ul of the PMN/test material the wells. Cyclosporin A (2.5 Lig/ml) was used as a positive combination was then spotted onto the membrane above each control, while untreated cells served as a negative control of the bottom wells (materials were tested in triplicate) and 65 (Untreated). One set of wells was not treated with PHA (Un the well plate was incubated for 1 hour at 37+2°C. and 5% treated-PHA). After the wells had been prepared, the plates CO. At the end of the incubation, the top of the chemotaxis were incubated for approximately 68 hours at 37+2° C. and US 8,932,652 B2 91 92 5% CO. After the incubation, 20 Jul of a 20:1 solution of TABLE 1.8 MTS:PMS (Promega) was added to each well and the 96-well plate was returned to the incubator for an additional 4 hours. Gamma Interferon ASSay The plate was then read at 490 nm using a plate reader. At the Treatment pg/ml end of the incubation period the cell culture supernatant was 1.5% LSPS 1116 109.7 assayed for IL-1C. and gamma interferon. O.S9/o LSPS 3924 - 425.8 Prior to the assay, all buffers and reagents were prepared O.1% LSPS 4626 - 305.5 according to the ELISA kit instructions (IL-1C.: Cayman 1.5% HSPS 997 - 159.7 Chemicals (catalog number 583301); Y-IFN: R&D Systems O.S90 HSPS 3509203.O 10 O.1% HSPS 4125 - 1314 (catalog number DIF50)) and allowed to reach room tempera 2.5 ugml Cyclosporin A 3259,326.8 ture. The 96-well plate was prepared by removing any Untreated 1107831S.O unneeded well strips and by rinsing the plate once with Wash Untreated -PHA 83 - 7.O Buffer followed by blotting it dry. Next, a series of IL-1C. standards was prepared ranging from 0 to 250 pg/ml and 100 ul of each of these standards was dispensed into two wells 15 Example 27 (duplicates) in the 96-well plate. Subsequently, 50 ul of each sample+50 ul of culture media was added to additional wells (the samples were diluted to bring their IL-1C. levels within Antiinflammatory Properties of Materials from Microal the range of the standard curve), followed by the addition of gae 100 ul of acetylcholinesterase:Interleukin-1C. FAB' Conju When presented with certainantigens, lymphocytes, which gate Solution. After reserving two empty wells to serve as are the main cell type in PBMCs, respond by proliferating. blanks, the 96-well plate was incubated overnight at 2-8°C. This massive proliferation reaction forms the initial step in On the following day the wells were aspirated and washed 5-6 the immune response of this cell type. In vitro were used times with Wash Buffer. After the last wash was removed 200 methods to study human lymphocyte proliferation using the ul of fresh Ellman's Reagent was added to each well. The 25 antigen phytohemagglutinin (PHA) to stimulate a prolifera plate was incubated at room temperature with periodic checks tion response. The ability of microalgal materials to inhibit of the absorbance readings (405 nm) using a plate reader. this proliferation was tested. A series of gamma interferon standards was prepared rang Heparinized whole blood (20-30 ml) was collected from a ing from 15.6 pg/ml to 1000 pg/ml using human gamma healthy human donor, layered over a density gradient (Histo interferon. Next, an ELISA microplate was prepared by 30 paque 1077 and Histopaque 1119) and centrifuged at 400 g removing any unneeded strips from the plate frame. 100 ul of for 30 min. The PBMC rich fraction was removed and washed Assay Diluent RD1F was added to each well, followed by the twice in with 5.0 ml RPMI-1640 without phenol red supple addition of 100 ul of sample (diluted in culture media if necessary to bring the gamma interferon values within the mented with 5% heat-treated fetal calf serum (RPMI-FCS). range of the standard curve) or 100 ul of standard per well. 35 After washing, the cells were resuspended in RPMI-FCS at a The microplate was covered and allowed to incubate for final density of 1x10 cells/ml. 2+0.25 hours at room temperature. After the incubation the Fifty microliters of PBMC cells were added to wells in a plate was aspirated and washed three times with 400 ul of 96-well plate (each treatment was tested in triplicate). Next, wash buffer. After the last wash was removed 200 ul of anti 500 of RPMI-FCS supplemented with 2 g/ml PHA and the gamma interferon antibody/horseradish peroxidase conju 40 respective test materials was added to the wells. Cyclosporin gate solution was added to each well and the plate was incu A (2.5ug/ml) was used as a positive control, while untreated bated for 1+0.25 hour at room temperature. The wells were cells served as a negative control (Untreated). One set of wells aspirated and washed again as described above. After the last wash was removed 200ul of horseradish peroxidase substrate was not treated with PHA and served as an index of non Solution (hydrogen peroxide+tetramethylbenzidine as a chro stimulated proliferation (Untreated-PHA); all samples but magen) was added to each well and the plate was incubated 45 this sample were treated with PHA. After the wells had been for 20+5 minutes at room temperature. After the incubation prepared, the plates were incubated for approximately 68 50 ul of stop solution (2 N sulfuric acid) was added to each hours at 37+2°C. and 5% CO. After the incubation, 20 Jul of well and the plate was read using a microplate reader at 450 a 20:1 solution of MTS:PMS (Promega) was added to each . well and the 96-well plate was returned to the incubator for an As demonstrated by Tables 17 and 18 and FIGS. 13(a) and 50 additional 4 hours. The plate was then read at 490 nm using a 13(b), all materials tested at all concentrations reduced plate reader. gamma interferon and IL1-C. by human PBMC cells. The mean absorbance of the three wells of PBMC not TABLE 17 treated with PHA was determined and used to represent non 55 stimulated proliferation. The absorbance values for the IL-1C Assay PBMC from each treatment group were also then determined. A stimulated proliferation index for each treatment was cal Treatment pg/ml culated using the following equations: (Mean absorbance of 1.5% LSPS 689 693 treatment with PHA)/(Mean Absorbance without PHA))x O.S9/o LSPS 1530 - 79.7 1OO O.1% LSPS 1715 69.3 60 1.5% HSPS 56338.5 The results for the PBMC proliferation assay are presented O.S90 HSPS 146299.9 in Table 19 and FIG. 18. The values are expressed as a stimu O.1% HSPS 1663 541 lation index, using the untreated PBMC not exposed to PHA 2.5 ugml Cyclosporin A 1561 77.5 Untreated 24O2 - 146.2 to represent 100%. As can be seen in Table 19 and FIG. 18, all Untreated -PHA 1512, 88.8 65 materials tested reduced PBMC proliferation compared to PHA stimulated cells that were untreated by the test materi als. US 8,932,652 B2 93 94 TABLE 19 A Bicinchoninic Acid assay (Pierce, Inc., BCA Protein Assay Kit, catalogi23227) Fifty volumes of Reagent A Proliferation ASSay (Bicinchoninic Acid BCA was combined with 1 volume of Treatment Stimulation Index Reagent B (4% (w/v) CuSO-5 HO) in a 15-ml centrifuge tube. For the assay, proteins can reduce Cu(II) to Cu(I) in a 1.5% LSPS 3216.0 O.S9/o LSPS 106 6.9 concentration dependent manner. BCA can then react with O.1% LSPS 111 12.0 the Cu(I) to form a purple colored complex with a maximum 1.5% HSPS SO 13.6 absorbance at 562 nm. Two hundred microliters of this com O.S90 HSPS 117 14.4 bined reagent was dispensed into a 96-well plate. Next, 10 ul O.1% HSPS 126-344 10 2.5 ugml Cyclosporin A 123 - 6.5 of each of the standards was added into their respective wells Untreated 183 13.9 (standards were made using 2 mg/ml bovine serum albumin Untreated -PHA 1OO 5.2 dissolved in PBS, and then making a series of 50% dilutions down to 0.0078 mg/ml). Next, 10 ul of 2x elastase buffer 15 (used as a blank) or cell lysate sample was added. The plate Example 28 was covered and incubated at 37+2°C. for 30+5 minutes. At the end of the incubation the absorbance of each well was Elastase Inhibition measured at 540 nm using a microplate reader. Human dermal fibroblasts were cultured and used as a After measuring the protein concentration of the lysate, a Source of the elastase enzyme. This enzyme was be partially Small sample was obtained and the elastase activity of the purified from the fibroblasts by lysing the cells in an elastase lysate was determined at 100%, 50% and 25% concentrations buffer and retaining the soluble portion of the lysate. Portions using 2x elastase buffer to make the dilutions. To determine of this fibroblast lysate were then be incubated with test the activity, 100 ul of the three different lysate concentrations materials a synthetic elastase Substrate. Suc-(Alas)-p-Nitroa was loaded into a 96 well plate (each concentration tested in niline (STANA). Elastase acts upon this substrate to release 25 triplicate), while 100 ul of 2x Elastase buffer was added to p-nitroaniline, which is detected with a spectrophotometer by three additional wells to serve as blanks. Next, 100 ul of measuring the absorbance at a wavelength of 405 nm. An deionized water was added to all wells and the 96-well plate inhibition of the elastase enzyme is identified by a decrease in will be allowed to incubate with the samples for 15 minutes at the amount of released p-nitro aniline when compared to 37+2° C. Next, 4 ul of STANA (62.5 mM Suc-(Ala)-p- uninhibited enzyme. 30 Nitroaniline in DMSO) was added to each well, and the Test material BM was supplied as a powder. 100 mg of BM 96-well plate was returned to the incubator for 1 hour. After was combined with either 5 ml of ethanol or 5 ml of ultrapure the incubation the well plate was read at 405 nm using a water in 15 ml centrifuge tubes. After combining, the mix microplate reader. The mean absorbance for each concentra tures were Vortexed, then placed onto a rocking platform for tion of the lysate was plotted versus its respective concentra approximately 30 minutes at room temperature, and then 35 tion. These values were used to estimate a concentration that centrifuged at 1,000 RPM for 5 minutes. The supernatants will produce a spectrophotometric measurement of 0.4 to 0.5 were then used at the final concentrations listed in Table 20. using a linear regression analysis of the best-fit line through The two additional materials used in this study, PS53 and all three (100%, 50%, and 25%) data points. The stored fibro PS133, were supplied as thick, viscous solutions (3%). These blast lysates was diluted to the appropriate concentration to materials were also used at the final concentrations listed in 40 elicit the desired level of activity. the results section. Test materials were prepared at 2x their final desired con Human neonatal dermal fibroblasts were obtained and cul centration in deionized water. If solvents were present in the tured per the vendor's specifications using sterile technique at test materials then appropriate solvent controls were also all times (Cascade Biologics, catalog #C-004-5C). The cells prepared. 100 ul of distilled water served as a negative control were seeded in a 75-cm flask and grown in Fibroblast 45 while 100 ul of 0.2 mM Phosphoramidon served as a positive Growth Medium (FGM: DMEM supplemented with 2% control. After all of the wells had been prepared the well plate FBS, 5 ng/ml human recombinant growth factor, 5 ug/ml was sealed with an adhesive cover and incubated for 15 min insulin, 50lug/ml gentamicin, and 0.5ug/ml Amphotericin-B) utes at 37+2° C. to allow the inhibitors time to interact with and subcultured until a sufficient number of cells had been elastase. After this preliminary 15 minute incubation, 4 p. 1 of grOWn. 50 STANA was added; the plate was resealed and then incubated After washing the cells twice with PBS, approximately 7.5 for 1 hour at 37+2°C. At the end of the incubation the plate ml of PBS was added to each culture flask and the cells were was read at 405 nm using a microplate reader. detached using a cell Scraper. The detached cells were trans ferred to a 15 ml centrifuge tube and the flask was rinsed with TABLE 20 a second application of 7.5 ml of PBS, which was also trans 55 ferred to the 15 ml tube. After centrifuging the tube for 5 Anti- Elastase Assay minutes at 1,200 RPM (4°C.), the supernatant was removed Treatment Percent Inhibition and discarded while the pellet was resuspended in 2 ml of ice 10% BM water extraction 59 cold 2x elastase buffer (400 mM Tris-HCl pH 8.0, 0.2% 5% BMwater extraction 40 Triton X-100) and sonicated 3x for 10 seconds or until the 60 10% BM Ethanol extraction 28 lysate became clear. The lysed cells were centrifuged at 2.200 5% BM Ethanol extraction 16 RPM for 10 minutes (4°C.) to remove any cellular debris. The 196 LSPS 29 Supernatants from all of the preparations were pooled into a O.S9/o LSPS 23 196 LSPS 31 single container, mixed, and then aliquoted into 2 ml portions O.S90 HSPS 19 and stored at -75+5° C. until used. One of the aliquots was 65 100 IM Phosphoramidon 94 used to determine the protein concentration (BCA Protein Assay) and elastase activity level of the batch. US 8,932,652 B2 95 96 Example 29 TABLE 21-continued Microalgal materials were tested to assess their ability to Superoxide dismutases relieve redness and pain associated with Sunburn. Informed Genbank accession number consent was obtained from human Subjects. A 1.5% prepara 5 ZP 01641300 ZP O1638301 tion of polysaccharide from Porphyridium cruentum, purified ZP 01637188 essentially as described in example 2, was formulated with EAX24391 1% Germaben II and 0.15% sodium EDTA. A patch of arm EAX23794 EAX23720 skin was exposed to UV radiation from a Solar Ultraviolet 10 EAX23627 Simulator model 14S with a dose controller system model EAX2O859 DCS-1 and a 150 watt xenon arc lamp #1122200 (Hanovia) EAX19390 EAX16596 having a continuous emission spectrum in the UV-B range of CAL93139 290-320 nanometers. The dose was tested on each subject YP 914326 prior to the experiment to determine the minimal erythema 15 YP 7474.24 dose (MED) necessary to achieve a level 2 response in each ABIS9459 ZP O1610569 Subject. Erythema was scored by a trained evaluator using the ZP 01605216 following scale: 0 no reaction, +-minimal erythema: ZP O1600343 1=slight erythema; 2 moderate erythema; 3-marked ZP O1584,712 erythema with or without edema: 4-severe erythema with or ZP O1581863 ZP O1581157 without edema. Subjects were then exposed to a UVB dose ZP O1575777 sufficient to generate a level 2 erythema at three sites on the ZP O1569848 skin. One site was treated with the polysaccharide before and ZP O1559998 after irradiation. One site was treated with polysaccharide EAWO1367 EAWO1065 only after irradiation. One site was left untreated as a control. 25 EAV972.74 After 4, 24, 48,72, and 96 hours, follow-up examination was EAV95856 performed. Polysaccharide was applied once daily to the two EAV80568 treated sites following daily examinations and was also EAV73624 treated once per day at home by each subject. EAV73531 Mean erythema scores were the same on all three sites EAV7O130 30 EAV66456 tested through the 4 and 24 hour evaluation points. At the 48 EAV61854 hour evaluation the mean erythema score decreased 5% on ZP O1532079 the site treated pre and post irradiation and stayed the same on ZP O1516088 the other two sites. At the 72 hour evaluation the mean EAV26209 erythema scores decreased by 30% on both sites treated with YP 845889 the polysaccharide and decreased 13% on the untreated site. 35 YP 822355 YP 843115 At the 96 hour evaluation the mean erythema scores YP 836186 decreased by 40% on both sites treated with the polysaccha ABK17454 ride and decreased 22% on the untreated site. There was no edema observed at any time on any site. The polysaccharide treatment was associated with a clinically significant 40 decrease in erythema compared to no treatment. TABLE 22 All references cited herein, including patents, patent appli Beta-caroteine hydroxylases cations, and publications, are hereby incorporated by refer Genbank accession number ence in their entireties, whether previously specifically incor porated or not. 45 P 682690 P 172377 P 440788 TABLE 21 P. 475340 P. 475340 Superoxide dismutases AB7S708 Genbank accession number 50 P 01084736 P 01080915 CAA42737 P 01123496 CAA43859 BB27O16 AAA29934 P 895.643 BAAO26SS P 896386 NP 625295 55 BB34062 AHOO42OO P 292.794 CAC14367 AA P99312 YP 001003721 P 01006041 ABM60577 BB49299 CAMO87SS P 848964 YP 966270 60 P 0104.0435 YP 963716 P O49051 ABM37237 P 457405 ABM35060 AAT386.25 ABM33234 ABM33141 NP 407488 NP 405922 65 Having now fully described this invention, it will be appre YP 93.2026 ciated by those skilled in the art that the same can be per formed within a wide range of equivalent parameters, con US 8,932,652 B2 97 98 centrations, and conditions without departing from the spirit to cover any variations, uses, or adaptations of the invention and scope of the invention and without undue experimenta following, in general, the principles of the invention and tion. including Such departures from the present disclosure as While this invention has been described in connection with come within known or customary practice within the art to specific embodiments thereof, it will be understood that it is which the invention pertains and as may be applied to the capable of further modifications. This application is intended essential features hereinbefore set forth.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 4 O
<21 Os SEQ ID NO 1 &211s LENGTH: 253 &212s. TYPE: DNA <213> ORGANISM: Chlamydomonas reinhardtii <4 OOs SEQUENCE: 1 cgcttagaag atttcgataa gg.cgc.ca.gala ggagcgcagc caaaccagga tigatgtttga 60
tgggg tattt gagcacttgc aaccctt at C cqqaagc.ccc ctggcc caca aaggct aggc 12O gccalatgcaa gCagttcgca toagcc cct ggagcggtgc cct cct gata aaccggc.cag 18O
gggggctatgttctt tactt ttttacaaga gaagt cactic aa catc.ttaa acggtc.ttaa 24 O
gaagttctatic cq9 253
<21 Os SEQ ID NO 2 &211s LENGTH: 312 &212s. TYPE: DNA <213> ORGANISM: Chlamydomonas reinhardtii
<4 OOs SEQUENCE: 2 Ctttcttgcg ct atgacact tccagcaaaa gg tagggcgg gctg.cgaga C ggct tcc.cgg 60 cgctgcatgc aacaccgatg atgct tcgac ccc.ccgaagc tic ctitcgggg Ctgcatgggc 12O gct Cogatgc cgctic caggg cagogctgt ttaaatagoc aggc.ccc.cga ttgcaaagac 18O
attatagcga gctaccaaag ccatatt caa acaccitagat cactaccact tctacacagg 24 O ccact cqagc titgtgat cqc acticcgctaa gggggggcct ct tcct ctitc gttt cagt ca 3 OO
caa.ccc.gcaa ac 312
<21 Os SEQ ID NO 3 &211s LENGTH: 356 &212s. TYPE: DNA <213> ORGANISM: Chlorella
<4 OOs SEQUENCE: 3 cggggat.cgc agggcatggg cattaaaaga actt tatgga at Caaaaatc ttagtgaatt 60 to caccacag gtatatagt c tt caggacgc taacgatgat at caacgatt gtat caaagg 12O ttatcgtttg aggcact cat at Cagg tagt ttctacacag aaacttgaac aacgcc tiggg 18O
aaaagat colt gagcatagta acttatatac tagcagatgt td taacgatg ctittatatga 24 O
atatgaatta gcacaacgac aactacaaaa acaacttgat gaatttgacg aagatgggta 3 OO
tgattitttitt caggcacgta taaatacatt agat cogtcg acctgcagoc aagctt 356
<21 Os SEQ ID NO 4 &211s LENGTH: 2O7 &212s. TYPE: DNA <213> ORGANISM: Chlorella
<4 OOs SEQUENCE: 4 cc.cggggat C at Caaag.ca actgcc.gcat tcgaaacttic gactgcctcg ttataaaggt 60 tag tigaaagc cattg tatgt tattact gag titatttaatt tagcttgctt aaatgctitat 12O US 8,932,652 B2 99 100 - Continued cgtgttgata tdataaatga caaatgatac gotgitat caa catct caaaa gattaatacg 18O aagat.ccgt.c gacctgcagc caa.gctt 2O7
<210s, SEQ ID NO 5 &211s LENGTH: 277 &212s. TYPE: DNA <213s ORGANISM: Chlorella
<4 OOs, SEQUENCE: 5 cc.cggggatc. tcg tattgc gggacttittg agcatttitcc agaacggatt gcc.gggacgt. 6 O at actgaacc ticcagt ccct ttgct cqtcq tattt cocat aatatacata tacactattt 12 O taattattta caccggttgt togctgagtga tacaatgcaa attcc ct coa ccgaggagga 18O tcqcgaactg. tccaaatgtc. ttctittctgc agctic catac ggagt cqtta ggaaacatt c 24 O acttaattat aggat.ccgtc gacctgcago caa.gctt 277
<210s, SEQ ID NO 6 &211s LENGTH: 489 &212s. TYPE: DNA <213> ORGANISM: Rhodella reticulata
<4 OOs, SEQUENCE: 6 tttittataga t catccaatt atttittt cat tagatattgt at atcaataa tittgg catat 6 O gttttgtagt atacgggitta t datattgca atatatgtac alacattggta atttittggac 12 O ttacatatat atcaattata t caatgacaa totaatatat tdgttgatag atcaataaac 18O atctittaata agatctgtta aaattcaaat atagactittctgt attataa gtagttittct 24 O tat attacta tagacgtaga acgatcaaaa aaaaataaat atgga catga cittgattcaa 3OO tatggalagac gggg tatgag aaat atcgtg ttgcact caa tatagaattg acg tatttitt 360 aatgcagtgc ccgittatata ttgcgtaa.ca aagattaaaa gtatattata tattataata 42O c tagtag acc agcaaatata aaattatgct gaaacaataa taccctittaa agttittaagg 48O agcc ttitt c 489
<210s, SEQ ID NO 7 &211s LENGTH: 543 &212s. TYPE: DNA <213> ORGANISM: Porphyridium
<4 OO > SEQUENCE: 7 attatttaac aattggaaac ttagttaatt agggtaaatt at attaa.ccc titatgaacca 6 O aaataatttg gtttcaaaaa aaactaactt atgaattaaa attgaaatat tittctacatc 12 O ataataattt taattictaaa tagaattitta gataagggat citaagataac aaaaaaatca 18O atttaagtaa taaagaaaat gtgattacaa aatttittgat attaalactat agtatttaca 24 O aatt attatc aaaaattact tat coatttg aggaaaagac togaac ctitta aac at atttg 3OO tittatgcgat tittagat cat t caagttagc gagctgitatgaaatgaaagt tt catgtaca 360 gttcttaagt agagatgitat atatgttaat agaaat atta tittgcatcga citataat caa 42O ttctgaagac ttcaaaataa aacctgttat acgtgctata citagagatgg ttgatgaaat 48O aaatcaacca gg tatt atta cagactgaac tdaactaaaa aaatt catat aatttagcgt. 54 O act 543
<210s, SEQ ID NO 8
US 8,932,652 B2 103 104 - Continued tcqt cacc 1148
<210s, SEQ ID NO 10 &211s LENGTH: 587 212. TYPE : PRT <213> ORGANISM: Porphyridium sp.
<4 OOs, SEQUENCE: 10
Met Thr His Ile Glu Ser Asn Tyr Glin Glu Glin Thir Gly Ala Phe 1. 5 1O 15
Ala Luell Luell Asp Ser Lell Wall Arg His Lys Wall His Ile Phe Gly 25
Pro Gly Gly Ala Ile Lell Pro Ile Asp Glu Lell Tyr Trp 35 4 O 45
Glu Glu Glin Gly Tyr Ile Lys His Ile Luell Wall Arg His Glu Glin Gly SO 55 6 O
Ala Ala His Ala Ala Asp Gly Tyr Ala Arg Ala Thir Gly Glu Wall Gly 65 70
Wall Phe Ala Thir Ser Gly Pro Gly Ala Thir Asn Lell Wall Thir Gly 85 90 95
Ile Ala Thir Ala His Met Asp Ser Ile Pro Ile Wall Ile Ile Thir Gly 105 11 O
Glin Wall Gly Arg Ser Phe Ile Gly Thir Asp Ala Phe Glin Glu Wall Asp 115 12 O 125
Ile Phe Gly Ile Thir Lell Pro Ile Wall His Ser Wall Ile Arg 13 O 135 14 O
Asp Pro Arg Asp Ile Pro Arg Ile Wall Ala Glu Ala Phe Ser Ile Ala 145 150 155 160
Glin Arg Pro Gly Pro Wall Luell Ile Asp Wall Pro Asp Wall 1.65 17O 17s
Gly Luell Thir Phe Glu Tyr Glin Tyr Wall ASn Pro Gly Glu Ala Arg 18O 185 19 O
Ile Pro Phe Asp Lell Wall Ala Pro Ser Ser Arg Glin Ile Ile
His Ser Glin Lell Ile Glin Glu Ala Asn Glin Pro Lell Luell Tyr Wall 21 O 215 22O
Gly Gly Ala Ile Thir Ser Gly Ala His Asp Lell Ile Luell 225 23 O 235 24 O
Wall Asn Lys Ile Pro Ile Thir Thir Thir Lell Met Gly Lys Gly 245 250 255
Ile Ile Asp Glu Glin Asn Pro Luell Ala Luell Gly Met Lell Gly Met His 26 O 265 27 O
Gly Thir Ala Ala Asn Phe Ala Wall Ser Glu Asp Luell Luell Ile 27s 28O 285
Thir Luell Gly Ala Arg Phe Asp Asp Arg Wall Thir Gly Lys Luell Asp Glu 29 O 295 3 OO
Phe Ala Asn Ala Lys Wall Ile His Wall Asp Ile Asp Pro Ala Glu 3. OS 310 315
Wall Gly Asn Arg Ile Pro Glin Wall Ala Ile Wall Gly Asp Ile Ser 3.25 330 335
Lell Wall Luell Glu Glin Trp Lell Luell Tyr Luell Asp Arg Asn Luell Glin Luell 34 O 345 35. O
Asp Asp Ser His Lell Arg Ser Trp His Glu Arg Ile Phe Arg Trp Arg 355 360 365 US 8,932,652 B2 105 106 - Continued
Glin Glu Tyr Pro Lell Ile Wall Pro Luell Wall Glin Thir Luell Ser Pro 37 O 375
Glin Glu Ile Ile Ala Asn Ile Ser Glin Ile Met Pro Asp Ala Phe 385 390 395 4 OO
Ser Thir Asp Wall Gly Glin His Glin Met Trp Ala Ala Glin Phe Wall Lys 4 OS 415
Thir Luell Pro Arg Arg Trp Lell Ser Ser Ser Gly Lell Gly Thir Met Gly 425 43 O
Gly Luell Pro Ala Ala Ile Gly Ala Ile Ala Tyr Pro Glu Ser 435 44 O 445
Pro Wall Wall Cys Ile Thir Gly Asp Ser Ser Phe Glin Met Asn Ile Glin 450 45.5 460
Glu Luell Gly Thir Ile Ala Glin Tyr Luell Asp Ile Ile Ile Ile 465 470
Ile Asn Asn Trp Glin Gly Met Wall Arg Glin Ser Glin Ala Phe 485 490 495
Gly Ala Arg Tyr Ser His Ser Arg Met Glu Asp Ala Pro Asn SOO 505
Phe Wall Ala Luell Ala Ser Phe Gly Ile Asp Gly Ser Ile Ser 515
Thir Arg Glin Glu Met Asp Ser Luell Phe Asn Thir Ile 53 O 535 54 O
Gly Pro Met Wall Ile Asp Wall Ile Glu Asp Asn Tyr 5.45 550 555 560
Pro Met Wall Ala Pro Gly Ser Asn Ala Glin Met Gly Luell Asp 565 st O sts
Ser Asn Asn Glu Ile Ile Ile Glu 58O 585
<210s, SEQ ID NO 11 &211s LENGTH: 129 212. TYPE : PRT &213s ORGANISM: Streptoalloteichus hindustanus
<4 OOs, SEQUENCE: 11
Met Ala Arg Met Ala Lell Thir Ser Ala Wall Pro Wall Luell Thir Ala 1. 5 15
Arg Asp Wall Ala Gly Ala Wall Glu Phe Trp Thir Asp Arg Luell Gly Phe 25
Ser Arg Asp Phe Wall Glu Asp Asp Phe Ala Gly Wall Wall Arg Asp Asp 35 4 O 45
Wall Thir Luell Phe Ile Ser Ala Wall Glin Asp Glin Asp Glin Wall Wall Pro SO 55 6 O
Asp Asn Thir Luell Ala Trp Wall Trp Wall Arg Gly Lell Asp Glu Luell Tyr 65 70
Ala Glu Trp Ser Glu Wall Wall Ser Thir Asn Phe Arg Asp Ala Ser Gly 85 90 95
Pro Ala Met Thir Glu Ile Gly Glu Glin Pro Trp Gly Arg Glu Phe Ala 1OO 105 11 O
Lell Arg Asp Pro Ala Gly Asn Cys Wall His Phe Wall Ala Glu Glu Glin 115 12 O 125
Asp
<210s, SEQ ID NO 12 US 8,932,652 B2 107 108 - Continued
&211s LENGTH: 144 212. TYPE: PRT <213> ORGANISM: Haloarcula hispanica
<4 OOs, SEQUENCE: 12 Gly Tyr Val Asin Gly Lieu. Glu Ser Ala Glu Glu Thir Lieu Ala Glu Asn 1. 5 1O 15 Arg Glu Ser Gly Asp Phe Gly Ser Ser Ala Ala Ala Met Gly Asn Val 2O 25 3O Thr His Asn Gly Cys Gly His Tyr Lieu. His Thr Lieu Phe Trp Glu Asn 35 4 O 45 Met Asp Pro Asn Gly Gly Gly Glu Pro Glu Gly Glu Lieu. Lieu. Asp Arg SO 55 6 O Ile Glu Glu Asp Phe Gly Ser Tyr Glu Gly Trp Lys Gly Glu Phe Glu 65 70 7s 8O Ala Ala Ala Ser Ala Ala Gly Gly Trp Ala Lieu. Lieu Val Tyr Asp Pro 85 90 95 Val Ala Lys Glin Lieu. Arg Asn Val Pro Val Asp Llys His Asp Glin Gly 1OO 105 11 O Ala Lieu. Trp Gly Ser His Pro Ile Lieu Ala Lieu. Asp Val Trp Glu. His 115 12 O 125 Ser Tyr Tyr Tyr Asp Tyr Gly Pro Ala Arg Gly Asp Phe Ile Asp Ala 13 O 135 14 O
<210s, SEQ ID NO 13 &211s LENGTH: 154 212. TYPE PRT <213> ORGANISM: Homo sapiens
<4 OOs, SEQUENCE: 13 Met Ala Thir Lys Ala Val Cys Val Lieu Lys Gly Asp Gly Pro Val Glin 1. 5 1O 15 Gly Ile Ile Asin Phe Glu Gln Lys Glu Ser Asn Gly Pro Wall Lys Val 2O 25 3O Trp Gly Ser Ile Lys Gly Lieu. Thr Glu Gly Lieu. His Gly Phe His Val 35 4 O 45 His Glu Phe Gly Asp Asn Thr Ala Gly Cys Thir Ser Ala Gly Pro His SO 55 6 O Phe Asn Pro Lieu. Ser Arg Llys His Gly Gly Pro Lys Asp Glu Glu Arg 65 70 7s 8O His Val Gly Asp Lieu. Gly Asn Val Thir Ala Asp Lys Asp Gly Val Ala 85 90 95 Asp Wal Ser Ile Glu Asp Ser Val Ile Ser Lieu. Ser Gly Asp His Cys 1OO 105 11 O Ile Ile Gly Arg Thr Lieu Val Val His Glu Lys Ala Asp Asp Lieu. Gly 115 12 O 125 Lys Gly Gly Asn. Glu Glu Ser Thr Lys Thr Gly Asn Ala Gly Ser Arg 13 O 135 14 O
Lieu Ala Cys Gly Val Ile Gly Ile Ala Glin 145 150
<210s, SEQ ID NO 14 &211s LENGTH: 711 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Superoxide Dismutase-Polysaccharide binding protein fusion AAV485 90-NP OOO445 US 8,932,652 B2 109 110 - Continued
<4 OOs, SEQUENCE: 14
Met Ala Arg Met Wall Wall Ala Ala Wall Ala Wall Met Ala Wall Luell Ser 1. 5 15
Wall Ala Luell Ala Glin Phe Ile Pro Asp Wall Asp Ile Thir Trp Wall 25
Pro Met Thir Luell Thir Wall Glin Asn Luell Ser Ile Phe Thir Gly Pro Asn 35 4 O 45
Glin Phe Gly Arg Gly Ile Pro Ser Pro Ser Ala Ile Gly Gly Gly Asn SO 55 6 O
Gly Luell Asp Ile Wall Gly Gly Gly Gly Ser Luell Ile Ser Pro Thir 65 70
Gly Gly Glin Wall Glin Ser Arg Gly Ser ASn Asn Phe Gly Asn Glin 85 90 95
Wall Ala Phe Thir Wall Arg Asn Gly ASn Asn Glu Ser Asp Phe 105 11 O
Ala Thir Wall Phe Wall Gly Gly Thir Thir Pro Ser Phe Wall Ile Wall Gly 115 12 O 125
Asp Ser Thir Glu Asn Glu Wall Ser Phe Trp Thir Asn Asn Wall Wall 13 O 135 14 O
Wall Asn Ser Glin Gly Phe Ile Pro Pro Asn Gly Asn Ser Ala Gly Gly 145 150 155 160
Asn Ser Glin Thir Phe Wall Asn Gly Ile Thir Gly Thir Ala Gly Ala 1.65 17O 17s
Pro Wall Gly Gly Thr Wall Ile Arg Gln Wall Ser Ala Trp Arg Glu Ile 18O 185 19 O
Phe Asn Thir Ala Gly Asn Wall Ser Phe Gly Lell Wall Wall Arg 195
Gly Thir Gly Asn Glin Gly Lell Wall Glin Gly Wall Glu Asp Gly 21 O 215
Wall Ala Ile Asp Ser Asn Gly Ser Phe Ala Ile Ser Gly Ser Pro 225 23 O 235 24 O
Ala Wall Asn Asn Ala Pro Gly Phe Gly Lys ASn Phe Ala Ala Ala Arg 245 250 255
Thir Gly Asn Phe Phe Ala Wall Ser Ser Glu Ser Gly Wall Ile Wall Met 26 O 265 27 O
Ser Ile Pro Wall Asp Asn Ala Gly Thir Luell Ser Phe Ser Wall Ala 285
Thir Ile Thir Pro Gly Ala Gly Arg Wall Ser Gly Wall Ser Luell Ala 29 O 295 3 OO
Glin Asp Asn Glu Phe Tyr Ala Ala Wall Gly Ile Pro Gly Ala Gly Pro 3. OS 310 315
Gly Glu Wall Arg Ile Arg Luell Asp Gly Gly Gly Ala Thir Thir Luell 3.25 330 335
Wall Glin Thir Luell Ser Pro Pro Asp Asp Ile Pro Glu Lell Pro Ile Wall 34 O 345 35. O
Ala Asn Glin Arg Phe Gly Glu Met Wall Arg Phe Gly Ala Asn Ser Glu 355 360 365
Thir Asn Wall Ala Wall Gly Ser Pro Gly Ala Ala Glu Gly Luell 37 O 375 38O
Ala Luell Phe Thir Ala Glu Pro Gly Luell Thir Pro Asn Asp Pro Asp 385 390 395 4 OO
Glu Gly Luell Luell Thir Lell Lell Ala Ser ASn Ser Ser Glu Ile Pro US 8,932,652 B2 111 112 - Continued
4 OS 415
Ala Asn Gly Gly Lieu. Gly Glu Phe Met Thir Ala Ser Asn Cys Arg Glin 42O 425 43 O
Phe Val Phe Gly Glu Pro Ser Wall Asp Ser Wall Wall Thir Phe Luell Ala 435 44 O 445
Ser Ile Gly Ala Tyr Tyr Glu Asp Cys Thr Cys Glu Arg Glu Asn 450 45.5 460
Ile Phe Asp Glin Gly Ile Met Phe Pro Wall Pro Asn Phe Pro Gly Glu 465 470 48O
Ser Pro Thr Thr Cys Arg Ser Ser Ile Tyr Glu Phe Arg Phe Asn 485 490 495
Lieu Met Glu Gly Ala Pro Ser Ile Cys Thr Tyr Ser Glu Arg Pro Thir SOO 505
Tyr Glu Trp Thr Glu Glu Wall Wall Asp Pro Asp Asn Thir Pro Glu 515 525
Lieu Val Ser Arg Ile Glin Arg Arg Luell Ser Glin Ser Asn Phe Glin 53 O 535 54 O
Asp Tyr Val Thr Lieu Gln Wall Wall Gly Ala Gly Ala Gly Met Ala Thir 5.45 550 555 560
Lys Ala Val Cys Val Lieu. Gly Asp Gly Pro Wall Glin Gly Ile Ile 565 st O sts
Asn Phe Glu Glin Lys Glu Ser Asn Gly Pro Wall Wall Trp Gly Ser 58O 585 59 O
Ile Lys Gly Lieu. Thr Glu Gly Luell His Gly Phe His Wall His Glu Phe 595 605
Gly Asp Asn. Thir Ala Gly Cys Thir Ser Ala Gly Pro His Phe Asn Pro 610 615
Lieu. Ser Arg Llys His Gly Gly Pro Asp Glu Glu Arg His Wall Gly 625 630 635 64 O
Asp Lieu. Gly Asn Val Thr Ala Asp Asp Gly Wall Ala Asp Wall Ser 645 650 655
Ile Glu Asp Ser Val Ile Ser Luell Ser Gly Asp His Ile Ile Gly 660 665 67 O
Arg Thr Lieu Val Val His Glu Lys Ala Asp Asp Lell Gly Gly Gly 675 685
Asn Glu Glu Ser Thr Lys Thir Gly Asn Ala Gly Ser Arg Luell Ala 69 O. 695 7 OO
Gly Val Ile Gly Ile Ala Glin 7 Os 71O
<210s, SEQ ID NO 15 &211s LENGTH: 552 212. TYPE: PRT <213> ORGANISM: Porphyridium
<4 OOs, SEQUENCE: 15
Met Ala Arg Met Val Val Ala Ala Wall Ala Wall Met Ala Wall Luell Ser 1. 5 1O 15
Wall Ala Lieu Ala Glin Phe Ile Pro Asp Val Asp Ile Thir Trp Wall 2O 25 3O
Pro Met Thir Lieu. Thir Wall Glin Asn Luell Ser Ile Phe Thir Gly Pro Asn 35 4 O 45
Glin Phe Gly Arg Gly Ile Pro Ser Pro Ser Ala Ile Gly Gly Gly Asn SO 55 6 O US 8,932,652 B2 113 114 - Continued
Gly Luell Asp Ile Wall Gly Gly Gly Gly Ser Luell Tyr Ile Ser Pro Thir 65 70
Gly Gly Glin Wall Glin Ser Arg Gly Ser ASn Asn Phe Gly Asn Glin 85 90 95
Wall Ala Phe Thir Wall Arg Asn Gly ASn Asn Glu Ser Asp Phe 105 11 O
Ala Thir Wall Phe Wall Gly Gly Thir Thir Pro Ser Phe Wall Ile Wall Gly 115 12 O 125
Asp Ser Thir Glu Asn Glu Wall Ser Phe Trp Thir Asn Asn Wall Wall 13 O 135 14 O
Wall Asn Ser Glin Gly Phe Ile Pro Pro Asn Gly Asn Ser Ala Gly Gly 145 150 155 160
Asn Ser Glin Thir Phe Wall Asn Gly Ile Thir Gly Thir Ala Gly Ala 1.65 17O 17s
Pro Wall Gly Gly Thir Wall Ile Arg Glin Wall Ser Ala Trp Arg Glu Ile 18O 185 19 O
Phe Asn Thir Ala Gly Asn Wall Ser Phe Gly Lell Wall Wall Arg 195
Gly Thir Gly Asn Glin Gly Lell Wall Glin Gly Wall Glu Asp Gly 21 O 215
Wall Ala Ile Asp Ser Asn Gly Ser Phe Ala Ile Ser Gly Ser Pro 225 23 O 235 24 O
Ala Wall Asn Asn Ala Pro Gly Phe Gly Lys ASn Phe Ala Ala Ala Arg 245 250 255
Thr Gly Asn Phe Phe Ala Wall Ser Ser Glu Ser Gly Wall Ile Wall Met 26 O 265 27 O
Ser Ile Pro Wall Asp Asn Ala Gly Thir Luell Ser Phe Ser Wall Ala 285
Thir Ile Thir Pro Gly Ala Gly Arg Wall Ser Gly Wall Ser Luell Ala 29 O 295 3 OO
Glin Asp Asn Glu Phe Tyr Ala Ala Wall Gly Ile Pro Gly Ala Gly Pro 3. OS 310 315
Gly Glu Wall Arg Ile Arg Luell Asp Gly Gly Gly Ala Thir Thir Luell 3.25 330 335
Wall Glin Thir Luell Ser Pro Pro Asp Asp Ile Pro Glu Lell Pro Ile Wall 34 O 345 35. O
Ala Asn Glin Arg Phe Gly Glu Met Wall Arg Phe Gly Ala Asn Ser Glu 355 360 365
Thir Asn Wall Ala Wall Gly Ser Pro Gly Ala Ala Glu Gly Luell 37 O 375
Ala Luell Phe Thir Ala Glu Pro Gly Luell Thir Pro Asn Asp Pro Asp 385 390 395 4 OO
Glu Gly Luell Luell Thir Lell Lell Ala Ser ASn Ser Ser Glu Ile Pro 4 OS 41O 415
Ala Asn Gly Gly Lell Gly Glu Phe Met Thir Ala Ser Asn Cys Arg Glin 425 43 O
Phe Wall Phe Gly Glu Pro Ser Wall Asp Ser Wall Wall Thir Phe Luell Ala 435 44 O 445
Ser Ile Gly Ala Tyr Glu Asp Thir Cys Glu Arg Glu Asn 450 45.5 460
Ile Phe Asp Glin Gly Ile Met Phe Pro Wall Pro Asn Phe Pro Gly Glu 465 470 47s 48O
Ser Pro Thir Thir Cys Arg Ser Ser Ile Glu Phe Arg Phe Asn US 8,932,652 B2 115 116 - Continued
485 490 495
Lell Met Glu Gly Ala Pro Ser Ile Cys Thr Tyr Ser Glu Arg Pro Thir SOO 505 51O
Tyr Glu Trp Thr Glu Glu Wall Wall Asp Pro Asp Asn Thr Pro Cys Glu 515 52O 525
Lieu Wal Ser Arg Ile Glin Arg Arg Lieu. Ser Glin Ser Asn Cys Phe Glin 53 O 535 54 O
Asp Tyr Wall Thir Lieu. Glin Wall Wall 5.45 550
<210s, SEQ ID NO 16 &211s LENGTH: 2O7 &212s. TYPE: DNA <213> ORGANISM: Chlamydomonas reinhardtii
<4 OOs, SEQUENCE: 16 gccagaagga gcgcagccala accaggatga tigtttgatgg ggt atttgag cacttgcaac 6 O c ctitat cogg aagcc.ccctg gcc cacaaag gct aggcgcc aatgcaa.gca gttcgcatgc 12 O agcc cctgga gcggtgcc ct cctgataaac cqgcc agggg gcc tatgttc titt actttitt 18O tacaagagaa gtcact caac at Cttaa.
SEO ID NO 17 LENGTH: 706 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Superoxide Dismutase-Polysaccharide binding protein fusion AAV485 90-NP OOO445 <4 OOs, SEQUENCE: 17
Met Ala Thir Lys Ala Wall Wall Luell Lys Gly Asp Gly Pro Wall Glin 1. 5 1O 15
Gly Ile Ile Asn. Phe Glu Glin Glu Ser ASn Gly Pro Wall Llys Val 2O 25
Trp Gly Ser Ile Llys Gly Lell Thir Glu Gly Luell His Gly Phe His Wall 35 4 O 45
His Glu Phe Gly Asp Asn Thir Ala Gly Thir Ser Ala Gly Pro His SO 55 6 O
Phe Asn Pro Luell Ser Arg His Gly Gly Pro Asp Glu Glu Arg 65 70 8O
His Wall Gly Asp Lieu. Gly Asn Wall Thir Ala Asp Asp Gly Wall Ala 85 90 95
Asp Wall Ser Ile Glu Asp Ser Wall Ile Ser Luell Ser Gly Asp His Cys 1OO 105 11 O
Ile Ile Gly Arg Thr Lell Wall Wall His Glu Lys Ala Asp Asp Lieu. Gly 115 12 O 125
Gly Gly Asn. Glu Glu Ser Thir Thir Gly Asn Ala Gly Ser Arg 13 O 135 14 O
Lell Ala Gly Val Ile Gly Ile Ala Glin Met Ala Arg Met Wall Wall 145 150 155 160
Ala Ala Wall Ala Wall Met Ala Wall Luell Ser Wall Ala Lell Ala Glin Phe 1.65 17O 17s
Ile Pro Asp Val Asp Ile Thir Trp Lys Wall Pro Met Thir Luell Thir Wall 18O 185 19 O
Glin Asn Luell Ser Ile Phe Thir Gly Pro Asn Glin Phe Gly Arg Gly Ile 195 2OO 2O5 US 8,932,652 B2 117 118 - Continued
Pro Ser Pro Ser Ala Ile Gly Gly Gly Asn Gly Lell Asp Ile Wall Gly 21 O 215
Gly Gly Gly Ser Lell Tyr Ile Ser Pro Thir Gly Gly Glin Wall Glin Tyr 225 23 O 235 24 O
Ser Arg Gly Ser Asn Asn Phe Gly Asn Glin Wall Ala Phe Thir Arg Wall 245 250 255
Arg Asn Gly Asn Asn Glu Ser Asp Phe Ala Thir Wall Phe Wall Gly 26 O 265 27 O
Gly Thir Thir Pro Ser Phe Wall Ile Wall Gly Asp Ser Thir Glu Asn Glu 27s 285
Wall Ser Phe Trp Thir Asn Asn Wall Wall Wall Asn Ser Glin Gly Phe 29 O 295 3 OO
Ile Pro Pro Asn Gly Asn Ser Ala Gly Gly ASn Ser Glin Tyr Thir Phe 3. OS 310 315
Wall Asn Gly Ile Thir Gly Thir Ala Gly Ala Pro Wall Gly Gly Thir Wall 3.25 330 335
Ile Arg Glin Wall Ser Ala Trp Arg Glu Ile Phe Asn Thir Ala Gly Asn 34 O 345 35. O
Wall Lys Ser Phe Gly Lell Wall Wall Arg Gly Thir Gly Asn Glin Gly 355 360 365
Lell Wall Glin Gly Wall Glu Tyr Asp Gly Tyr Wall Ala Ile Asp Ser Asn 37 O 375
Gly Ser Phe Ala Ile Ser Gly Tyr Ser Pro Ala Wall Asn Asn Ala Pro 385 390 395 4 OO
Gly Phe Gly Asn Phe Ala Ala Ala Arg Thir Gly Asn Phe Phe Ala 4 OS 415
Wall Ser Ser Glu Ser Gly Wall Ile Wall Met Ser Ile Pro Wall Asp Asn 425 43 O
Ala Gly Cys Thir Lell Ser Phe Ser Wall Ala Tyr Thir Ile Thir Pro Gly 435 44 O 445
Ala Gly Arg Wall Ser Gly Wall Ser Luell Ala Glin Asp Asn Glu Phe Tyr 450 45.5 460
Ala Ala Wall Gly Ile Pro Gly Ala Gly Pro Gly Glu Wall Arg Ile Tyr 465 470
Arg Luell Asp Gly Gly Gly Ala Thir Thir Luell Wall Glin Thir Luell Ser Pro 485 490 495
Pro Asp Asp Ile Pro Glu Lell Pro Ile Wall Ala Asn Glin Arg Phe Gly SOO 505
Glu Met Wall Arg Phe Gly Ala Asn Ser Glu Thir Asn Tyr Wall Ala Wall 515 525
Gly Ser Pro Gly Tyr Ala Ala Glu Gly Luell Ala Lell Phe Thir Ala 53 O 535 54 O
Glu Pro Gly Luell Thir Pro Asn Asp Pro Asp Glu Gly Lell Luell Thir Luell 5.45 550 555 560
Lell Ala Ser Asn Ser Ser Glu Ile Pro Ala Asn Gly Gly Luell Gly 565 st O sts
Glu Phe Met Thir Ala Ser Asn Arg Glin Phe Wall Phe Gly Glu Pro 58O 585 59 O
Ser Wall Asp Ser Wall Wall Thir Phe Luell Ala Ser Ile Gly Ala Tyr 595 6OO 605
Glu Asp Thir Glu Arg Glu Asn Ile Phe Asp Glin Gly Ile 610 615 62O US 8,932,652 B2 119 120 - Continued
Met Phe Pro Wall Pro Asn Phe Pro Gly Glu Ser Pro Thir Thir Cys Arg 625 630 635 64 O
Ser Ser Ile Glu Phe Arg Phe Asn Cys Luell Met Glu Gly Ala Pro 645 650 655
Ser Ile Thir Tyr Ser Glu Arg Pro Thir Tyr Glu Trp Thir Glu Glu 660 665 67 O
Wall Wall Asp Pro Asp Asn Thir Pro Glu Luell Wall Ser Arg Ile Glin 675 68O 685
Arg Arg Luell Ser Glin Ser Asn Phe Glin Asp Tyr Wall Thir Luell Glin 69 O. 695 7 OO
Wall Wall 7 Os
SEQ ID NO 18 LENGTH: 763 TYPE : PRT ORGANISM: Chlamydomonas reinhardtii
< 4 OOs SEQUENCE: 18
Met Lieu. Ala Ser Thir Thir Pro Gly Wall Arg Glin Wall Ala Gly 1. 5 15
Arg Thir Wall Ala Wall Pro Ser Ser Luell Wall Ala Pro Wall Ala Wall Ala 25
Arg Ser Luell Gly Lell Ala Pro Tyr Wall Pro Wall Glu Pro Ser Ala 35 4 O 45
Ala Luell Pro Ala Cys Glin Glin Pro Ser Gly Arg Arg His Wall Glin Thir 50 55 60
Ala Ala Thir Luell Arg Ala Asp Asn Pro Ser Ser Wall Glin Luell Wall 65 70
His Glin Asn Gly Lys Gly Met Wall Ile Ile Ala Ala Gly Ile 85 90 95
Gly Gly Luell Wall Lell Ala Wall Ala Luell Luell Glin Phe Glin Wall 105 11 O
Glin Wall Phe Glu Arg Asp Lell Thir Ala Ile Arg Gly Gly Tyr 115 12 O
Arg Gly Pro Ile Glin Wall Glin Ser Asn Ala Luell Ala Luell Glu Ala 13 O 135 14 O
Ile Asp Pro Glu Wall Ala Ala Glu Wall Luell Arg Glu Ile Thir 145 150 155 160
Gly Asp Arg Ile Asn Gly Lell Asp Gly Luell Thir Glu Trp Tyr 1.65 17O 17s
Wall Phe Asp Thir Phe His Pro Ala Wall Ser Luell Pro Wall 18O 185 19 O
Thir Arg Wall Ile Ser Arg Lell Thir Luell Glin Glin Ile Lell Ala Ala 195 2OO 2O5
Wall Glu Arg Gly Gly Pro Gly Thir Ile Glin Asn Gly Asn Wall 21 O 215 22O
Thir Glu Phe Thir Glu Arg Arg Asn Asp Thir Thir Gly Asn Asn Glu Wall 225 23 O 235 24 O
Thir Wall Glin Luell Glu Asp Gly Arg Thir Phe Ala Ala Asp Wall Luell Wall 245 250 255
Gly Ala Asp Gly Ile Trp Ser Ile Arg Glin Lell Ile Gly Glu 26 O 265 27 O
Thir Ala Asn Tyr Ser Gly Tyr Thir Cys Thir Gly Ile Ser Asp 27s 28O 285 US 8,932,652 B2 121 122 - Continued
Phe Thir Pro Ala Asp Ile Asp Ile Wall Gly Arg Wall Phe Luell Gly 29 O 295 3 OO
Asn Gly Glin Phe Wall Ser Ser Asp Wall Gly Asn Gly Met Glin 3. OS 310 315
Trp Gly Phe His Glu Pro Ser Gly Gly Thir Asp Pro Glu Gly 3.25 330 335
Ser Arg Ala Arg Lell Lell Glin Ile Phe Gly His Trp Asn Asp Asn 34 O 345 35. O
Wall Wall Asp Luell Ile Ala Thir Pro Glu Glu Asp Wall Luell Arg Arg 355 360 365
Asp Ile Phe Asp Arg Pro Pro Ile Phe Thir Trp Ser Gly Arg Wall 37 O 375
Ala Luell Luell Gly Asp Ser Ala His Ala Met Glin Pro Asn Luell Gly Glin 385 390 395 4 OO
Gly Gly Met Ala Ile Glu Asp Ala Tyr Glu Lell Ala Ile Asp Luell 4 OS 415
Ser Arg Ala Wall Ser Asp Ala Gly Asn Ala Ala Ala Wall Asp Wall 425 43 O
Glu Gly Wall Luell Arg Ser Glin Asp Ser Arg Ile Lell Arg Wall Ser 435 44 O 445
Ala Ile His Gly Met Ala Gly Met Ala Ala Phe Met Ala Ser Thir 450 45.5 460
Lys Luell Gly Glu Gly Trp Ser Trp Wall Glu Gly Luell Arg 465 470
Ile Pro His Pro Gly Arg Wall Wall Gly Arg Luell Wall Met Luell Luell Thir 485 490 495
Met Pro Ser Wall Lell Glu Trp Wall Luell Gly Gly Asn Thir Asp His Wall SOO 505
Ala Pro His Arg Thir Ser Cys Ser Luell Gly Asp Lys Pro Ala 515 525
Phe Pro Glu Ser Arg Phe Pro Glu Phe Met ASn Asn Asp Ala Ser Ile 53 O 535 54 O
Ile Arg Ser Ser His Ala Asp Trp Luell Luell Wall Ala Glu Arg Asp Ala 5.45 550 555 560
Ala Thir Ala Ala Ala Ala Asn Wall Asn Ala Ala Thir Gly Ser Ser Ala 565 st O sts
Ala Ala Ala Ala Ala Ala Asp Wall Asn Ser Ser Glin Cys Gly 585 59 O
Ile Met Ala Asp Ser Ala Ala Luell Wall Gly Arg Cys Gly Ala Thir 595 605
Ser Arg Pro Ala Lell Ala Wall Asp Asp Wall His Wall Ala Glu Ser His 610 615 62O
Ala Glin Wall Trp Arg Gly Lell Ala Gly Luell Pro Pro Ser Ser Ser Ser 625 630 635 64 O
Ala Ser Thir Ala Ala Ala Ser Ala Ser Ala Ala Ser Ser Ala Ala Ser 645 650 655
Gly Thir Ala Ser Thir Lell Gly Ser Ser Glu Gly Trp Luell Arg Asp 660 665 67 O
Lell Gly Ser Gly Arg Gly Thir Trp Wall Asn Gly Arg Luell Pro Asp 675 685
Gly Ala Thir Wall Glin Lell Trp Pro Gly Asp Ala Wall Glu Phe Gly Arg 69 O. 695 7 OO US 8,932,652 B2 123 124 - Continued
His Pro Ser His Glu Wall Phe Llys Val Lys Met Gln His Wall Thr Luell 7 Os 71s 72O
Arg Ser Asp Glu Lieu. Ser Gly Glin Ala Tyr Thir Thir Lieu Met Wall Gly 72 73 O 73
Ile Arg Asn. Asn Asp Wal Met Pro Glu Ser Arg Pro Asp Gly 740 74. 7 O
Gly Ser Glin Gln Pro Gly Arg Lieu Val Thr Ala 760
<210s, SEQ ID NO 19 &211s LENGTH: 524 212. TYPE : PRT <213> ORGANISM: Arabidopsis thaliana
<4 OOs, SEQUENCE: 19
Met Glu. Cys Val Gly Ala Arg Asn Phe Ala Ala Met Ala Wall Ser Thir 1. 5 1O 15
Phe Pro Ser Trp Ser Arg Arg Llys Phe Pro Wall Wall Lys Arg 25
Ser Arg Asn Ile Arg Phe Gly Lieu. Cys Ser Wall Arg Ala Ser Gly 35 4 O 45
Gly Gly Ser Ser Gly Ser Glu Ser Cys Val Ala Wall Arg Glu Asp Phe SO 55 6 O
Ala Asp Glu Glu Asp Phe Wall Lys Ala Gly Gly Ser Glu Ile Luell Phe 65 70
Wall Glin Met Glin Glin Asn Asp Met Asp Glu Glin Ser Luell Wall 85 90 95
Asp Luell Pro Pro Ile Ser Ile Gly Asp Gly Ala Lell Asp Luell Wall 105 11 O
Wall Ile Gly Gly Pro Ala Gly Lieu Ala Luell Ala Ala Glu Ser Ala 115 12 O 125
Luell Gly Luell Wall Gly Lieu. Ile Gly Pro Asp Lell Pro Phe Thir 13 O 135 14 O
Asn Asn Tyr Gly Wall Trp Glu Asp Glu Phe ASn Asp Lell Gly Luell Glin 145 150 155 160
Ile Glu His Wall Trp Arg Glu Thir Ile Wall Luell Asp Asp 1.65 17O 17s
Asp Pro Ile Thir Ile Gly Arg Ala Tyr Gly Arg Wall Ser Arg Arg 18O 185 19 O
Lell Luell His Glu Glu Lell Lell Arg Arg Cys Wall Glu Ser Gly Wall Ser 195 2OO 2O5
Luell Ser Ser Wall Asp Ser Ile Thr Glu Ala Ser Asp Gly Luell 21 O 215
Arg Luell Wall Ala Asp Asp Asn Asn. Wall Ile Pro Arg Luell Ala 225 23 O 235 24 O
Thir Wall Ala Ser Gly Ala Ala Ser Gly Lys Luell Lell Glin Glu Wall 245 250 255
Gly Gly Pro Arg Wall Wall Glin. Thir Ala Gly Wall Glu Wall Glu 26 O 265 27 O
Wall Glu Asn Ser Pro Asp Pro Asp Glin Met Wall Phe Met Asp 27s 28O 285
Arg Asp Thir Asn Glu Lys Val Arg Ser Luell Glu Ala Glu Tyr Pro 29 O 295 3 OO
Thir Phe Luell Ala Met Pro Met Thr Lys Ser Arg Lell Phe Phe Glu 3. OS 310 315 32O
US 8,932,652 B2 129 130 - Continued
Wall Ala Luell Ala Glin Phe Ile Pro Asp Wall Asp Ile Thir Trp Lys Wall 25
Pro Met Thir Luell Thir Wall Glin Asn Luell Ser Ile Phe Thir Gly Pro Asn 35 4 O 45
Glin Phe Gly Arg Gly Ile Pro Ser Pro Ser Ala Ile Gly Gly Gly Asn SO 55 6 O
Gly Luell Asp Ile Wall Gly Gly Gly Gly Ser Luell Tyr Ile Ser Pro Thir 65 70
Gly Gly Glin Wall Glin Ser Arg Gly Ser ASn Asn Phe Gly Asn Glin 85 90 95
Wall Ala Phe Thir Wall Arg Asn Gly ASn Asn Glu Ser Asp Phe 105 11 O
Ala Thir Wall Phe Wall Gly Gly Thir Thir Pro Ser Phe Wall Ile Wall Gly 115 12 O 125
Asp Ser Thir Glu Asn Glu Wall Ser Phe Trp Thir Asn Asn Wall Wall 13 O 135 14 O
Wall Asn Ser Glin Gly Phe Ile Pro Pro Asn Gly Asn Ser Ala Gly Gly 145 150 155 160
Asn Ser Glin Tyr Thir Phe Wall Asn Gly Ile Thir Gly Thir Ala Gly Ala 1.65 17O 17s
Pro Wall Gly Gly Thir Wall Ile Arg Glin Wall Ser Ala Trp Arg Glu Ile 18O 185 19 O
Phe Asn Thir Ala Gly Asn Wall Ser Phe Gly Lell Wall Wall Arg 195
Gly Thr Gly Asn Gln Gly Lieu Wall Gln Gly Wall Glu Tyr Asp Gly Tyr 21 O 215
Wall Ala Ile Asp Ser Asn Gly Ser Phe Ala Ile Ser Gly Tyr Ser Pro 225 23 O 235 24 O
Ala Wall Asn Asn Ala Pro Gly Phe Gly Lys ASn Phe Ala Ala Ala Arg 245 250 255
Thir Gly Asn Phe Phe Ala Wall Ser Ser Glu Ser Gly Wall Ile Wall Met 26 O 265 27 O
Ser Ile Pro Wall Asp Asn Ala Gly Cys Thir Luell Ser Phe Ser Wall Ala 285
Thir Ile Thir Pro Gly Ala Gly Arg Wall Ser Gly Wall Ser Luell Ala 29 O 295 3 OO
Glin Asp Asn Glu Phe Tyr Ala Ala Wall Gly Ile Pro Gly Ala Gly Pro 3. OS 310 315
Gly Glu Wall Arg Ile Arg Luell Asp Gly Gly Gly Ala Thir Thir Luell 3.25 330 335
Wall Glin Thir Luell Ser Pro Pro Asp Asp Ile Pro Glu Lell Pro Ile Wall 34 O 345 35. O
Ala Asn Glin Arg Phe Gly Glu Met Wall Arg Phe Gly Ala Asn Ser Glu 355 360 365
Thir Asn Tyr Wall Ala Wall Gly Ser Pro Gly Tyr Ala Ala Glu Gly Luell 37 O 375
Ala Luell Phe Thir Ala Glu Pro Gly Luell Thir Pro Asn Asp Pro Asp 385 390 395 4 OO
Glu Gly Luell Luell Thir Lell Lell Ala Ser ASn Ser Ser Glu Ile Pro 4 OS 41O 415
Ala Asn Gly Gly Lell Gly Glu Phe Met Thir Ala Ser Asn Cys Arg Glin 42O 425 43 O
Phe Wall Phe Gly Glu Pro Ser Wall Asp Ser Wall Wall Thir Phe Luell Ala US 8,932,652 B2 131 132 - Continued
435 44 O 445
Ser Ile Gly Ala Tyr Glu Asp Cys Thir Cys Glu Arg Glu Asn 450 45.5 460
Ile Phe Asp Glin Gly Ile Met Phe Pro Wall Pro Asn Phe Pro Gly Glu 465 470 47s
Ser Pro Thir Thir Cys Arg Ser Ser Ile Tyr Glu Phe Arg Phe Asn 485 490 495
Lell Met Glu Gly Ala Pro Ser Ile Cys Thir Ser Glu Arg Pro Thir SOO 505
Glu Trp Thir Glu Glu Wall Wall Asp Pro Asp Asn Thir Pro Glu 515 525
Lell Wall Ser Arg Ile Glin Arg Arg Luell Ser Glin Ser Asn Phe Glin 53 O 535 54 O
Asp Wall Thir Lell Glin Wall Wall Met Ile Arg Lell Gly Ala Pro Glin 5.45 550 555 560
Ser Luell Wall Luell Lell Thir Lell Luell Wall Ala Ala Wall Lell Arg Cys Glin 565 st O sts
Gly Glin Asp Wall Arg Glin Pro Gly Pro Gly Glin Gly Glu Pro 585 59 O
Gly Asp Ile Asp Ile Wall Gly Pro Gly Pro Pro Gly Pro Glin 595 605
Gly Pro Ala Gly Glu Glin Gly Pro Arg Gly Asp Arg Gly Asp Gly 610 615
Glu Gly Ala Pro Gly Pro Arg Gly Arg Asp Gly Glu Pro Gly Thir 625 630 635 64 O
Pro Gly Asn Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro 645 650 655
Gly Luell Gly Gly Asn Phe Ala Ala Glin Met Ala Gly Gly Phe Asp Glu 660 665 67 O
Ala Gly Gly Ala Glin Lell Gly Wall Met Glin Gly Pro Met Gly Pro 675 685
Met Gly Pro Arg Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Pro Glin 69 O. 695 7 OO
Gly Phe Gly Asn Pro Gly Glu Pro Gly Glu Pro Gly Wall Ser Gly 7 Os
Pro Met Pro Arg Gly Pro Pro Gly Pro Pro Gly Pro Gly Asp 72 73 O 73
Asp Gly Ala Gly Pro Gly Lys Ala Gly Glu Arg Gly Pro Pro 740 74. 7 O
Gly Pro Gly Ala Arg Gly Phe Pro Gly Thir Pro Gly Luell Pro Gly 760 765
Wall Lys His Arg Gly Tyr Pro Gly Luell Asp Gly Ala Gly Glu 770 775
Ala Gly Pro Gly Wall Gly Glu Ser Gly Ser Pro Gly Glu Asn 79 O 79.
Gly Ser Pro Gly Pro Met Gly Pro Arg Gly Luell Pro Gly Glu Arg Gly 805 810 815
Arg Thir Gly Pro Ala Gly Ala Ala Gly Ala Arg Gly Asn Asp Gly Glin 82O 825 83 O
Pro Gly Pro Ala Gly Pro Pro Gly Pro Wall Gly Pro Ala Gly Gly Pro 835 84 O 845
Gly Phe Pro Gly Ala Pro Gly Ala Gly Glu Ala Gly Pro Thir Gly 850 855 860 US 8,932,652 B2 133 134 - Continued
Ala Arg Gly Pro Glu Gly Ala Glin Gly Pro Arg Gly Glu Pro Gly Thir 865 87O 87s
Pro Gly Ser Pro Gly Pro Ala Gly Ala Ser Gly Asn Pro Gly Thr Asp 885 890 895
Gly Ile Pro Gly Ala Lys Gly Ser Ala Gly Ala Pro Gly Ile Gly 9 OO 905 91 O
Ala Pro Gly Phe Pro Gly Pro Arg Gly Pro Pro Gly Pro Gln Gly Ala 915 92 O 925
Thir Gly Pro Leu Gly Pro Lys Gly Glin Thr Gly Glu Pro Ala 93 O 935 94 O
Gly Phe Gly Glu Glin Gly Pro Gly Glu Pro Gly Pro Gly 945 950 955 96.O
Pro Glin Gly Ala Pro Gly Pro Ala Gly Glu Glu Gly Arg Ala 965
Arg Gly Glu Pro Gly Gly Val Gly Pro Ile Gly Pro Pro Gly Glu Arg 98O 985 99 O
Gly Ala Pro Gly Asn Arg Gly Phe Pro Gly Glin Asp Gly Lieu Ala Gly 995 1OOO
Pro Lys Gly Ala Pro G y Glu Arg G y Pro Ser G Ala O1O
Pro Ala Asn G y Asp Pro G y Arg Pro G Glu Pro O25
Lell Pro Ala Arg G y Lieu. Thr G Pro G Asp Ala O4 O
Pro Glin Llys Val G y Pro Ser G Pro G Glu Asp O55
Arg Pro Pro Pro G y Pro Glin G Arg G Glin Pro Of O
Wall Met Phe Pro G y Pro Lys G ASn G Glu Pro O85
Glu Lys G y Leu Pro G Pro G Luell Arg
Lell y Glu Thr G Ala G Pro Pro
Pro Pro Ala G Glin G Ala Pro
Pro Phe Glin G y Leu Pro G y Pro Pro G Pro Pro
Glu Llys Pro G y Asp Glin G y Val Pro G Glu Ala
Ala Lieu Val G y Glu Phe Pro
Glu Ser Pro G y Ala Glin G y Lieu. Glin G Pro Arg
Lell Pro Thir Pro G y Pro Ala Ser 2O5
Pro Pro Pro G y Ala Glin G y Pro Pro G Luell Glin 22O
Met Pro Glu Arg G y Ala Ala G y Ile Ala G Pro 235
Asp Asp Val G y Glu Lys G y Pro Glu G Ala Pro 250 255 US 8,932,652 B2 135 136 - Continued
Asp Gly Arg y Lieu. Thir Pro Ile y Pro Pro G 265 27 O
Pro Ala Asn y Glu Glu Wall Pro Pro 285
Pro Ser Ala Arg Ala Pro Glu Arg
Pro Pro Ala Phe Ala Pro Pro
Glin Pro Lys Glu Glin Glu Ala
Asp Ala Pro Pro Glin Pro Ser
Pro Glin Thir Wall Thir Pro
Ala Glin Pro Ala Thir Phe
Arg Wall Pro Ser Asn ASn
Pro Pro Pro Ser Asp Pro
Ala Asp Ser Pro Arg Ala Glu
Lell Pro Ala Pro Glu Glu
Asp Pro Ser Glu Pro Pro Pro Gln
Lell Glin Arg Wall Luell Pro Glin Arg
Glu Phe Pro Pro Ser Glu Pro
Ala Ser Asp Arg Pro Pro
Pro Pro Thir Pro Ala Glu Pro
Arg Ser Asp Pro Pro Arg Asp
Ala Wall Arg Glu Thir Ala Wall
Ala Pro Thir Pro Ser Pro Pro Ala 550
Pro Glin Arg Glu Ala Ala Glin 565
Pro Met Ser Ala Ala Arg Ile Glin 58O
Pro Glin Arg Lys Glu Ala Glu Pro 595
Glu Lel Arg Phe Thir Luell Glin 610
Lell Pro Pro y Pro Ser Asp Glin Ala Ser 625 63 O
Pro Ser y Pro Arg Pro Pro Pro Wall 64 O 645
Pro Ser Asp y Ala Asn Ile Pro Pro Ile US 8,932,652 B2 137 138 - Continued
1655 1660 1665
Pro Pro Gly Pro Arg Gly Arg Ser Gly Glu Thr Gly Pro Ala Gly 1670 1675 168O
Pro Pro Gly Asn Pro Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly 1685 1690 1695
Pro Gly Ile Asp Met Ser Ala Phe Ala Gly Lieu. Gly Pro Arg 17 OO 1705 1710
<210s, SEQ ID NO 23 &211s LENGTH: 1309 212. TYPE: PRT ORGANISM: Artificial Sequence 22 Os. FEATURE: OTHER INFORMATION: Glycoprotein-elastin fusion AAW48590 - NP OOO 492
<4 OOs, SEQUENCE: 23
Met Ala Arg Met Val Val Ala Ala Wall Ala Wall Met Ala Wall Lieu. Ser 1. 5 15
Wall Ala Lieu Ala Glin Phe Ile Pro Asp Wall Asp Ile Thir Trp Llys Val 2O 25 3O
Pro Met Thir Lieu. Thir Wall Glin Asn Luell Ser Ile Phe Thr Gly Pro Asn 35 4 O 45
Glin Phe Gly Arg Gly Ile Pro Ser Pro Ser Ala Ile Gly Gly Gly Asn SO 55 6 O
Gly Lieu. Asp Ile Val Gly Gly Gly Gly Ser Luell Tyr Ile Ser Pro Thr 65 70 8O
Gly Gly Glin Val Glin Tyr Ser Arg Gly Ser ASn Asn Phe Gly Asn Glin 85 90 95
Wall Ala Phe Thir Arg Val Arg Llys Asn Gly ASn Asn Glu Ser Asp Phe 1OO 105 11 O
Ala Thr Val Phe Val Gly Gly Thr Thir Pro Ser Phe Val Ile Val Gly 115 12 O 125
Asp Ser Thr Glu Asn. Glu Wal Ser Phe Trp Thir Asn Asn Llys Val Val 13 O 135 14 O
Wall Asn Ser Glin Gly Phe Ile Pro Pro Asn Gly Asn Ser Ala Gly Gly 145 150 155 160
Asn Ser Glin Tyr Thr Phe Val Asn Gly Ile Thir Gly. Thir Ala Gly Ala 1.65 17O 17s
Pro Val Gly Gly Thr Val Ile Arg Glin Wall Ser Ala Trp Arg Glu Ile 18O 185 19 O
Phe Asn. Thir Ala Gly Asn. Cys Val Ser Phe Gly Lieu Val Val Arg 195 2OO 2O5
Gly Thr Gly Asn Glin Gly Lieu Val Glin Gly Wall Glu Tyr Asp Gly Tyr 21 O 215 22O
Wall Ala Ile Asp Ser Asn Gly Ser Phe Ala Ile Ser Gly Tyr Ser Pro 225 23 O 235 24 O
Ala Val Asn. Asn Ala Pro Gly Phe Gly Lys ASn Phe Ala Ala Ala Arg 245 250 255
Thir Gly Asin Phe Phe Ala Val Ser Ser Glu Ser Gly Val Ile Val Met 26 O 265 27 O
Ser Ile Pro Val Asp Asn Ala Gly Thir Luell Ser Phe Ser Wall Ala 27s 28O 285
Thir Ile Thr Pro Gly Ala Gly Arg Wall Ser Gly Val Ser Lieu Ala 29 O 295 3 OO US 8,932,652 B2 139 140 - Continued
Glin Asp Asn Glu Phe Tyr Ala Ala Wall Gly Ile Pro Gly Ala Gly Pro 3. OS 310 315
Gly Glu Wall Arg Ile Arg Luell Asp Gly Gly Gly Ala Thir Thir Luell 3.25 330 335
Wall Glin Thir Luell Ser Pro Pro Asp Asp Ile Pro Glu Lell Pro Ile Wall 34 O 345 35. O
Ala Asn Glin Arg Phe Gly Glu Met Wall Arg Phe Gly Ala Asn Ser Glu 355 360 365
Thir Asn Wall Ala Wall Gly Ser Pro Gly Ala Ala Glu Gly Luell 37 O 375
Ala Luell Phe Thir Ala Glu Pro Gly Luell Thir Pro Asn Asp Pro Asp 385 390 395 4 OO
Glu Gly Luell Luell Thir Lell Lell Ala Ser ASn Ser Ser Glu Ile Pro 4 OS 415
Ala Asn Gly Gly Lell Gly Glu Phe Met Thir Ala Ser Asn Cys Arg Glin 425 43 O
Phe Wall Phe Gly Glu Pro Ser Wall Asp Ser Wall Wall Thir Phe Luell Ala 435 44 O 445
Ser Ile Gly Ala Tyr Glu Asp Thir Cys Glu Arg Glu Asn 450 45.5 460
Ile Phe Asp Glin Gly Ile Met Phe Pro Wall Pro Asn Phe Pro Gly Glu 465 470
Ser Pro Thir Thir Cys Arg Ser Ser Ile Tyr Glu Phe Arg Phe Asn 485 490 495
Lieu Met Glu Gly Ala Pro Ser Ile Cys Thr Ser Glu Arg Pro Thr SOO 505
Glu Trp Thir Glu Glu Wall Wall Asp Pro Asp Asn Thir Pro Glu 515 525
Lell Wall Ser Arg Ile Glin Arg Arg Luell Ser Glin Ser Asn Phe Glin 53 O 535 54 O
Asp Wall Thir Lell Glin Wall Wall Met Ala Gly Lell Thir Ala Ala Ala 5.45 550 555 560
Pro Arg Pro Gly Wall Lell Lell Luell Luell Luell Ser Lell His Pro Ser 565 st O sts
Arg Pro Gly Gly Wall Pro Gly Ala Ile Pro Gly Wall Pro Gly Gly 585 59 O
Wall Phe Tyr Pro Gly Ala Gly Luell Gly Ala Luell Gly Gly Ala Luell 595 605
Gly Pro Gly Gly Lys Pro Lell Pro Wall Pro Gly Luell Ala Gly 610 615
Ala Gly Luell Gly Ala Gly Lell Gly Ala Phe Pro Wall Thir Phe Pro 625 630 635 64 O
Gly Ala Luell Wall Pro Gly Gly Wall Ala Asp Ala Ala Ala Tyr 645 650 655
Ala Ala Ala Gly Ala Gly Luell Gly Gly Wall Pro Gly Wall Gly Gly 660 665 67 O
Lell Gly Wall Ser Ala Gly Ala Wall Wall Pro Glin Pro Gly Ala Gly Wall 675 685
Pro Gly Wall Pro Gly Wall Gly Luell Pro Gly Wall Pro Gly 69 O. 695 7 OO
Gly Wall Luell Pro Gly Ala Arg Phe Pro Gly Wall Gly Wall Luell Pro Gly 7 Os 71O
Wall Pro Thir Gly Ala Gly Wall Pro Lys Ala Pro Gly Wall Gly Gly US 8,932,652 B2 141 142 - Continued
72 73 O 73
Ala Phe Ala Gly Ile Pro Gly Wall Gly Pro Phe Gly Gly Pro Pro 740 74. 7 O
Gly Wall Pro Lieu. Gly Tyr Pro Lys Ala Pro Lell Pro Gly 765
Gly Luell Pro Tyr Thir Thir Llys Lieu Pro Tyr Gly Tyr Pro 770 775
Gly Gly Wall Ala Gly Ala Lys Ala Gly Tyr Pro Thir Thir 78s 79 O 79.
Gly Wall Gly Pro Glin Ala Ala Ala Ala Ala Ala Ala 805 810
Phe Gly Ala Gly Ala Wall Leu Pro Gly Wall Gly Ala 82O 825 83 O
Gly Wall Pro Gly Val Pro Ile Pro Gly Ile Gly Gly Ala 835 845
Gly Wall Gly Thir Pro Ala Ala Ala Ala Ala Ala Ala 850 860
Ala Ala Ala Gly Lieu Val Pro Gly Gly Pro Gly 865
Phe Gly Pro Gly Val Wall Wall Pro Gly Ala Gly Wall Pro Gly Wall 885 890 895
Gly Wall Pro Gly Ala Gly Pro Wall Wall Pro Gly Ala Gly Ile Pro 9 OO 905 91 O
Gly Ala Ala Wall Pro Gly Wall Wall Ser Pro Glu Ala Ala Ala Ala 915 920 925
Ala Ala Ala Ala Tyr Gly Ala Arg Pro Gly Wall Gly Wall Gly 93 O 935 94 O
Gly Ile Pro Thr Tyr Gly Wall Gly Ala Gly Gly Phe Pro Gly Phe Gly 945 950 955 96.O
Wall Gly Wall Gly Gly Ile Pro Gly Val Ala Gly Wall Pro Ser Wall Gly 965 97O 97.
Gly Wall Pro Gly Val Gly Gly Wall Pro Gly Val Gly Ile Ser Pro Glu 98O 985 99 O
Ala Glin Ala Ala Ala Ala Lys Ala Ala Lys Tyr Gly Wall Gly Thr 995
Pro Ala Ala Ala Ala Ala Lys Ala Ala Ala Lys Ala Ala Glin Phe O2O
Gly Luell Wall Pro Gly Val Gly Val Ala Pro Gly Wall Gly Wall Ala O25 O35
Pro Wall Gly Val Ala Pro Gly Wall Gly Lell Ala Pro Gly Wall OSO
Gly Wall Ala Pro Gly Val G y Val Ala Pro Gly Wall Gly Wall Ala O55 O65
Pro Ile Gly Pro y Val Ala Ala Ala Ser Ala
Ala Wall Ala Ala a Glin Luell Arg Ala Ala Ala Gly Lell O95
Gly Ile Pro Gly Lieu. Gly Wall Gly Wall Gly Wall Pro Gly 11 O
Lell Wall Gly Ala Gly Val Pro Gly Luell Gly Wall Gly Ala Gly 2O 125
Wall Pro Gly Phe Gly Ala G y Ala Asp Glu Gly Wall Arg Arg Ser 13 O 14 O
US 8,932,652 B2 147 148 - Continued <223> OTHER INFORMATION: GLP-forward primer <4 OOs, SEQUENCE: 29 acagcgctgt act coc 16
SEQ ID NO 3 O LENGTH: 23 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: GLUT1-forward primer
<4 OOs, SEQUENCE: 3 O tgctgatgat galacctgctg gcc 23
SEQ ID NO 31 LENGTH: 22 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: GLUT1-reverse primer
SEQUENCE: 31 agcacaatgc actggagcag cq 22
<21Os SEQ ID NO 32 <211 > LENGTH: 185 <212> TYPE: DNA <213> ORGANISM: Cauliflower Mosaic Wirus
< 4 OO SEQUENCE: 32 tittctic cata ataatgtgtgagtagttcCC agata aggga attagggitt C ctatagggitt 6 O tcqct catgt gttgagcata taagaaac cc ttagtatgta tttgt atttg taaaatactt 12 O citat caataa aatttctaat tcc taaaacc aaaatccagt actaaaatcc agat.ccc.ccg 18O aatta 185
<210s, SEQ ID NO 33 &211s LENGTH: 672 212. TYPE : PRT <213> ORGANISM: Cyanidioschyzon merolae
<4 OOs, SEQUENCE: 33
Met Ala Ser Thr Ala Pro Ala Asn Thir Ala Wall Ala Lell Asn Thir Luell 1. 5 1O 15
Asp Trp Wall Luell Wall Ile Wall Phe Ser Ala Lell Wall Luell Ala Ile 2O 25
Trp Luell Ser Thir Ser Ser Arg Ala Gly Ser Gly Thir Glin Ser 35 4 O 45
Pro Ala Ser Glu Met Phe Phe Lieu Ala Gly Arg Ser Thir Thir Phe SO 55 6 O
Phe Ala Wall Gly Ala Ser Lell Phe Met Ser ASn Ile Gly Ser Glu His 65 70
Phe Ile Ala Luell Ala Ala Ala Gly Ala Thr Ser Gly Lell Ala Wall Ala 85 90 95
Ser Phe Glu Trp Met Ala Ser Ile Phe Wall Gly Wall Wall Luell Gly Arg 105 11 O
Wall Phe Ala Pro Phe Lell Arg Asn. Ser Luell His Thir Wall Pro Lys 115 12 O 125
Phe Luell Glu Luell Arg Ala Ala Gly Ala Arg Arg His Ala Luell US 8,932,652 B2 149 150 - Continued
13 O 135 14 O
Ala Thir Ile Met Met Ala Ile Luell Thir Wall Ser Ala Thir Luell Tyr 145 150 155 160
Ser Gly Ala Ile Ile Lell Arg Wall Luell Luell Gly Trp Pro Wall Trp Phe 1.65 17O 17s
Ser Luell Ile Met Ile Lell Wall Luell Thir Thir Luell Tyr Thir Ser Luell Gly 18O 185 19 O
Gly Luell Arg Ala Wall Ile Trp Thir Glu Wall Luell Glin Ala Phe Wall Luell 195
Lell Ala Gly Gly Lell Ala Lell Ala Wall Arg Ser Lell Glin Ala Wall Gly 21 O 215
Ser Luell Ala Gly Lell Ser Glu Luell Luell Ala Ala Glin Asn Arg Arg Glin 225 23 O 235 24 O
Met Luell Asp Luell Lell Glin Trp Pro Ser Ser Thir Thir Pro Trp Wall Glu 245 250 255
Pro Trp Pro Gly Ile Ile Phe Gly Luell Pro Ala Lell Glu Wall Phe 26 O 265 27 O
Trp Cys Thir Asp Glin Wall Wall Wall Glin Arg Wall Lell Ser Ala 27s 285
Ser Glu Ala His Ala Arg Gly Gly Ser Luell Luell Cys Gly Phe Luell 29 O 295 3 OO
Thir Luell Wall Pro Phe Met Met Wall Ile Pro Gly Lell Ala Phe Luell 3. OS 310 315
Lell Phe Pro Glu Wall Ala Ala Asn Pro Asn Glin Ala Pro Thir Ala 325 330 335
Wall Ala Arg Luell Lell Pro His Gly Luell Luell Gly Lell Met Wall Ser Ala 34 O 345 35. O
Met Luell Ala Ala Lell Met Ser Ser Luell Ala Ser Thir Phe Asn Ser Thir 355 360 365
Ser Thir Wall Wall Wall Asp Phe Wall Ile Glu Cys Gly Luell Ser 37 O 375
Arg Luell Ser Asp Thir Lell Wall Luell Luell Gly Arg Ile Ala Asn Ile 385 390 395 4 OO
Wall Luell Ala Phe Ser Lell Ala Trp Ile Pro Ile Wall Glu Gly Met 4 OS 415
Gly Glu Glu Luell Tyr Phe Ile Glin Ser Wall Ile Ser Tyr Ile Ala 425 43 O
Pro Pro Ile Ala Wall Wall Phe Wall Ala Gly Ile Ala Trp Arg Arg Ala 435 44 O 445
Thir Ala Thir Gly Ala Lell Cys Thir Luell Luell Wall Gly Gly Ala Luell Gly 450 45.5 460
Lell Wall Arg Phe Wall Wall Glu Wall Ala Luell Arg Lell Ala His Arg Glu 465 470
Ala Pro Luell Gly Ala Lell Gly Ile Phe Phe Glin Ser Asn Phe Luell 485 490 495
Phe Ala Ile Phe Ser Trp Wall Phe Ser Ser Lell Lell Luell Wall Thir SOO 505 51O
Wall Ser Luell Phe Thir Glu Pro Pro Ser Glu Glin Glin Lell Gly Luell Luell 515 52O 525
Phe Glin Glu Ala Gly Ala Ser Gly Ser His Wall Arg Thir Thir Ala Gly 53 O 535 54 O
Asn Glin Ile Gly Glu Ala Ser Ser Glin Wall Wall Gly Ala Ser Arg 5.45 550 555 560 US 8,932,652 B2 151 152 - Continued
Wall Ala Asp Glu Pro Ser Ser Ser Asp Pro Ala Ala Arg Glin Glin 565 st O sts
Thir Wall Glu Luell Glu Ile Glu Ser Phe Ser Gly Thir Glu Asn Ser Asp 585 59 O
Glu Ala Phe Ser Wall Asp Pro Glu His Thir Ala Pro Ser Met 595 605
Wall Ala Ser Arg Asp Thir Lell Luell Ala Gly Glu Ala Ala Ser Glin Glu 610 615
Pro Luell Phe Ser Pro Glin Gly Glu Phe Ser Ala Ala Glin Glu Thir Phe 625 630 635 64 O
Ser Ser Ala Ala Pro Ser Arg Luell Thir Ser Ala Ala Lell Asp Wall Luell 645 650 655
Ser Wall Wall Luell Wall Ala Glu Ile Luell Ala Phe Ile Glin Phe Arg 660 665 67 O
SEQ ID NO 34 LENGTH: 541 TYPE : PRT ORGANISM: Cyanidioschyzon merolae arabinose
< 4 OOs SEQUENCE: 34
Met Glu. Thir Wall Thir Wall Arg Trp Phe Lell Ser Thir Phe Gly 1. 5 15
Ala Arg Pro Cys Lell Lell Ser Luell Luell Gly Gly Lell Luell Phe Gly 25
His Lieu Ala Wall Phe Ser Thr Wall Thr Thr Phe Arg Ser Phe Gln 35 4 O 45
Asp Trp Phe Gly Ser Trp Pro Ser Gly Glu Glin Wall Lell Luell Gly Ser SO 55 6 O
Tyr Phe Wall Gly Ala Phe Wall Gly Luell Tyr Glin Arg Wall Luell Pro 65 70
Phe Luell Ala His Gly Ala Gly Thir Ala Ala Trp Arg Arg Luell Trp 85 90 95
Lell Arg Trp Ser Ser Wall Phe Phe Cys Luell Gly Ser Gly Luell Pro Phe 105 11 O
Lell Ile Lys Arg Glu Arg Met Luell Thir Gly Arg Phe Glin Met Gly Ala 115 12 O 125
Phe Luell Wall Luell Lell Ile His Arg Luell Luell Ile Gly Ile Gly Ala Gly 13 O 135 14 O
Ile Wall Asn Wall Lell Gly Pro Ala Luell Luell Glu Wall Ala Pro Ser 145 150 155 160
Thir Ser Arg Gly Ala Phe Wall Phe Luell Tyr Glin Lell Ala Ile Thir Ile 1.65 17O 17s
Gly Ile Luell Met Ala Asn Lell Wall Asn Luell Ala Thir Gly His Glu Asp 18O 185 19 O
Wall His Arg Glin Wall Asp Pro Wall Ala Gly Gly Ala Gly Ile Asp Met 195
Arg Gly Asn Phe Lell Arg Pro Luell Arg Pro Lell Wall Pro Ala Ile 21 O 215
Lell Met Ile Gly Lell Lell Arg Glin Ser Ser Met Gly Wall Ser 225 23 O 235 24 O
Ala Arg Asp Ala Thir Ser Ala Asp Met Luell Glu Thir Gly Lys Pro 245 250 255
Met Ser Glin Arg Lys Glu Arg Glu His Ser Met Wall Ala Ser Ala Arg US 8,932,652 B2 153 154 - Continued
26 O 265 27 O
Ala Luell Arg Pro Asp Ala Ala Glu Ala Gly His Ser Phe Asn Wall Ile 27s 28O 285
Ser Arg Pro Ser Lell Glin Asn Glu Ser Luell Ser Lell Asp Wall Glu 29 O 295 3 OO
Ser Phe Ala Met Tyr Ala Wall Luell Ala Arg Gly Ser Ser Pro Thir Ala 3. OS 310 315
Arg Ser Arg Ala Ser Wall Trp Luell Luell Luell Arg Asp Pro Arg Ile Glin 3.25 330 335
Ile Met Ile Lell Glin Lell Luell Glin Glin Luell Thir Gly Ile Asn Wall 34 O 345 35. O
Wall Luell Wall Gly Wall Glin Ile Luell Glu Glin Wall Glin Ser Ser Ala 355 360 365
Met Gly Ser Ser Arg Arg Lell Ala Arg Luell Ser Gly Pro Luell Gly 37 O 375
Ala Wall Luell Luell Ser Wall Met Asn Wall Ile Ala Thir Lell Wall Ala Wall 385 390 395 4 OO
Gly Ile Ile Asp Arg Thir Arg Arg Lys Luell Lell Phe Ser Thir 4 OS 415
Pro Wall Luell Ala Ala His Luell Ala Luell Ala Arg Ala Thir Arg Ala 425 43 O
Glu Asn Gly Ser Ser Ser Wall Ala Phe Thir Gly Phe Lell Ala Luell Met 435 44 O 445
Lell Phe Wall Ala Wall Phe Ala Wall Ser His Gly Pro Lell Ala Wall Luell 450 45.5 460
Wall Ala Asn Glu Lell Phe Ser Pro Glu Ala Arg Ala Ser Ala Asn Ser 465 470
Ile Gly Met Wall Wall Asn Ala Wall Ala Thir Thir Ala Wall Ser Ile Gly 485 490 495
Phe Pro Luell Luell Glin Arg Glu Luell Phe Gly Ile Ala Gly Thir Phe Luell SOO 505
Phe Phe Ala Luell Ile Lell Met Gly Gly Glu Tyr Trp Lell Trp Arg 515 525
Lell Pro Glu Thir Glin Pro Thir Ser Ala Asp Ser Ser 53 O 535 54 O
<210s, SEQ ID NO 35 &211s LENGTH: 64 O 212. TYPE : PRT &213s ORGANISM: Galdieria sulphuraria
<4 OOs, SEQUENCE: 35
Met Val Glu Lys Ser Ser Asp Pro Glu Wall Pro Ser Lell Ser His His 1. 5 1O 15
Glu Ser Ser Ile Ser Ile Glu Glin Gly Asp Ala Ala Thir Ala Arg 2O 25
Glu Trp Ala Glin Asp Wall Asn Ser Thir Thir Thir Asn Thir Luell 35 4 O 45
Asn Pro Luell Ala Gly Lell Thir Arg Glu Glin Luell Lell Asn Asp Wall Glu SO 55 6 O
Ala Phe Ala Glu Lys Asp Luell Glu His Ile Lell Asp Asp Luell Arg 65 70 8O
Gly Ala Luell Wall Ala Glin Asp Pro Arg Glu Phe Glu Glin Met Asp 85 90 95 US 8,932,652 B2 155 156 - Continued
Ala Luell Thir Glu Ser Glu Glu Luell Luell Arg Arg Glu Lys Thir His 105 11 O
Arg Trp Ser Glin Pro Phe Met Met Phe Met Thir Ser Glu Ser Ser 115 12 O 125
Arg Tyr Pro Pro Thir Glu Phe Gly Phe Asn Pro Ala Glin Ser Ser 13 O 135 14 O
Wall Luell Asp Luell Lell Ser Arg Glu Trp Ile Arg Lell Luell Ser Thir 145 150 155 160
Wall Arg Arg Ser Met Ser Ser Ile Thir His Lell Ser Ala Lys 1.65
Glin Ser Arg Phe Tyr Phe Ala Glu Phe Asn Wall Thir Asp Thir Trp Met 18O 185 19 O
Glin Gly Luell Luell Asn Gly Ala Pro Luell Ser Ala Wall Ile Gly 195
Trp Thir Thir Ala Pro Lell Asn Arg Trp Phe Gly Arg Arg Gly Cys 21 O 215 22O
Ile Phe Ile Ser Cys Phe Ile Ser Phe Ala Ser Ser Phe Trp Met Ala 225 23 O 235 24 O
Ala Ala His Thir Trp Trp Asn Luell Luell Luell Gly Arg Phe Luell Luell Gly 245 250 255
Phe Ala Wall Gly Ala Ser Thir Thir Thir Pro Wall Gly Ala Glu 26 O 265 27 O
Ser Pro Ala Asn Ile Arg Gly Ala Luell Wall Met Met Trp Glin Met 27s 285
Trp Thr Ala Phe Gly Ile Met Lieu Gly Ile Ala Ser Wall Ala Phe 29 O 295 3 OO
Met Asp Wall Thir His Pro Thir Ile Pro Gly Phe Asn Trp Arg Luell Met 3. OS 310 315
Lell Gly Ser Thir Ala Ile Pro Pro Phe Phe Wall Ile Glin Wall Tyr 3.25 330 335
Phe Pro Glu Ser Pro Arg Trp Tyr Met Met Arg Asn Arg His 34 O 345 35. O
Asp Ala Tyr Ala Lell Lys Phe Arg Pro Ser Thir Phe Glin Ala 355 360 365
Ala Arg Asp Luell Tyr Ile His Ala Ala Luell Lys Wall Glu Glu Lys 37 O 375
Lell Arg Glu Gly Lys His Lell Phe Arg Glu Met Phe Thir Ile Pro Arg 385 390 395 4 OO
Asn Arg Arg Ala Ala Glin Ser Ser Phe Phe Wall Met Phe Met Glin Glin 4 OS 41O 415
Phe Gly Wall Asn Ala Ile Met Tyr Ser Ser Ser Met Phe Arg 425 43 O
Glu Ala Gly Phe Asp Thir Arg Met Ala Luell Ile Thir Ser Luell Gly Cys 435 44 O 445
Gly Ile Thir Asn Trp Ile Phe Ala Luell Pro Ala Wall Thir Ile Asp 450 45.5 460
Thir Phe Gly Arg Arg Asn Lell Luell Luell Thir Thir Phe Pro Luell Met Cys 465 470 47s 48O
Ile Phe Luell Luell Phe Thir Gly Phe Ser Phe Ile Pro Asp Glin Thir 485 490 495
Ser Arg Thir Ala Cys Wall Ala Thir Gly Ile Lell Phe Met Ile Wall SOO 505
Ser Pro Gly Glu Gly Pro Wall Pro Phe Thir Ser Ala Glu Ala