US 2005OOO3473A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0003473 A1 Bolten et al. (43) Pub. Date: Jan. 6, 2005
(54) ASPERGILLUS OCHRACEUS 11 ALPHA Publication Classification HYDROXYLASE AND OXDOREDUCTASE (51) Int. Cl." ...... C12P 33/06; C12P 33/08; C12N 9/02; C12N 1/16; CO7H 21/04; (75) Inventors: Suzanne L. Bolten, Kirkwood, MO C12N 15/74 (US); Robert A. Clayton, Foristell, MO (52) U.S. Cl...... 435/58; 435/59; 435/320.1; (US); Alan M. Easton, Maryland 435/189; 435/254.3; 536/23.2 Heights, MO (US); Leslie C. Engel, (57) ABSTRACT Des Peres, MO (US); Dean M. The present invention relates to a novel cytochrome P450 Messing, St. Louis, MO (US); John S. like enzyme (Aspergillus OchraceuS 11 alpha hydroxylase) Ng, Thousand Oaks, CA (US); Beverly and an oxidoreductase (Aspergillus Ochraceus oxidoreduc Reitz, Chesterfield, MO (US); Mark C. tase) isolated from cDNA library generated from the mRNA Walker, Chesterfield, MO (US); Ping of Aspergillus Ochraceus spores. When the cDNA encoding T. Wang, Chesterfield, MO (US) the 11 alpha hydroxylase was co-expressed in Spodoptera frugiperda (Sf-9) insect cells with the cDNA encoding human oxidoreductase as an electron donor, it Successfully Correspondence Address: catalyzed the conversion of the Steroid Substrate 4-andros PHARMACIA CORPORATION tene-3,17-dione (AD) to 11 alpha-hydroxy-AD as deter GLOBAL PATENT DEPARTMENT mined by HPLC analysis. The invention also relates to POST OFFICE BOX 1027 nucleic acid molecules associated with or derived from these ST. LOUIS, MO 63006 (US) cDNAS including complements, homologues and fragments thereof, and methods of using these nucleic acid molecules, (73) Assignee: Pharmacia Corporation to generate, for example, polypeptides and fragments thereof. The invention also relates to the generation of (21) Appl. No.: 10/900,856 antibodies that recognizes the A. OchraceuS 11 alpha hydroxylase and oxidoreductase and methods of using these (22) Filed: Jul. 28, 2004 antibodies to detect the presence of these native and recom Related U.S. Application Data binant polypeptides within unmodified and transformed host cells, respectively. The invention also provides methods of (62) Division of application No. 10/021,425, filed on Oct. expressing the Aspergillus 11 alpha hydroxylase gene Sepa 30, 2001. rately, or in combination with human or Aspergillus oxi doreductase, in heterologous host cells, to facilitate the (60) Provisional application No. 60/244,300, filed on Oct. bioconversion of steroid substrates to their 11 alpha 30, 2000. hydroxy-counterparts. Patent Application Publication Jan. 6, 2005 Sheet 1 of 25 US 2005/0003473 A1 Figure 1 - Nucleotide and protein sequence of Aspergillus ochraceus 11 alpha hydroxylase
A. tggaagtttt tacact tatt atgccggagc cgaaagattic tgagtcgagg ggttggggaa 60 caacactata agacctacaa coacttggat ttggtgaatt tacacgggca ttatcaaaac 2O agccacaagc tigacagctica ttatc atg CCC titC titc act ggg Ctt Ctg gCg 172 Met Pro Phe Phe Thr Gly Leu Leu Ala 5 att tac cat agt cto ata ctic gac aac Coa gtc. caa acc Ctg agc acc 220 Ile Tyr His Ser Leu Ile Leu Asp Asn Pro Val Glin Thr Leu Ser Thr O 5 20 25 att g to gta ttg gcg gca gcg tac togg Ctic gca acg Ctic Cag CCg agc 268 Ile Val Val Leu Ala Ala Ala Tyr Trp Leu Ala Thr Leu Gln Pro Ser 30 35 40 gac ctt cot gag ctgaat coc gCC aaa cca ttc gag titc acc aat Cgt 316 Asp Leu Pro Glu Leu Asn Pro Ala Lys Pro Phe Glu Phe Thr Asn Arg A5 50 SS cgt cgt gtt cat gag titt gtt gaa aat agt aag agc titg Ctt gct Cgg 364 Arg Arg Val His Glu Phe Val Glu Asn Ser Lys Ser Leu Leu Ala Arg 60 65 70 ggg agg gaa ttg cac ggg cac gag CCg tac aga Citc atg tot gaa togg 412 Gly Arg Glu Lieu. His Gly. His Glu Pro Tyr Arg Leu Met Ser Glu Trp 75 80 85 gga toc ttg att g to citg ccc cca gag togc gcc gaC gag Ctg cgC aac 460 Gly Ser Leu Ile Val Leu Pro Pro Glu Cys Ala Asp Glu Leu Arg Asn 90 95 OO O5 gac coa aga atg gac titt gag acg ccc acc acc gac gaC to C Cac gga 508 Asp Pro Arg Met Asp Phe Glu Thr Pro Thr Thr Asp Asp Ser His Gly 10 115 120 tat atc cct ggc titc gaC gct Ctc aac gca gaC ccg aac Citg act aaa 556 Tyr. Ile Pro Gly Phe Asp Ala Leu Asn Ala Asp Pro Asn Leu Thir Lys 125 130 3S gtg g to acc aag tac citc aca aaa gCattgaac aag Ctt act gct cog 604 Val Val Thr Lys Tyr Leu Thr Lys Ala Leu Asn Lys Leu Thr Ala Pro 14 O 145 SO atc. tcg cat gaa gog toc atc gcc atg aaa gCg gtg Ctg ggit gac gat 652 Ile Ser His Glu Ala Ser Ile Ala Met Lys Ala Val Leu Gly Asp Asp 155 160 1.65 cca gat tgg cgt gag atc tac coa gcc aga gaC titg ct C Cag Ctic gtC 700 Pro Asp Trp Arg Glu Ile Tyr Pro Ala Arg Asp Leu Leu Gln Leu Val 70 75 18O 185 gcc cgg atg tog aca aga gtg titc Ctt ggC gag gaa atg tgc aat aac 748 Ala Arg Met Ser Thr Arg Val Phe Leu Gly Glu Glu Met Cys Asn Asn 190 195 200 cag gat tgg atc caa acc toa toa caa tac gog gcc Ctt gcc titC ggt 796 Glin Asp Trp Ile Gln Thr Ser Ser Glin Tyr Ala Ala Leu Ala Phe Gly 2O5 210 215 g to ggt gac aag Ctt aga at a tac CCg aga atg at C aga CCg ata gta 84.4 Val Gly Asp Lys Leu Arg Ile Tyr Pro Arg Met Ile Arg Pro Ile Val 220 225 230 cat tgg titc atg cca toc tdt togg gag Ctg cgc cga ticg Ctg Cga Cgc 892 His Trp Phe Met Pro Ser Cys Trp Glu Leu Arg Arg Ser Leu Arg Arg 235 24 O 245 tgc cga cag att Ct c acg ccg tac att Cac aaa cgc aag to C Ctg aag 940 Cys Arg Glin Ile Leu Thr Pro Tyr Ile His Lys Arg Lys Ser Leu Lys 250 255 260 265 ggg acc acg gac gag cag ggc aag coc ctt atg titt gat gat tcc ac 988 Gly Thr Thr Asp Giu Gln Giy Lys Pro Leu Met Phe Asp Asp Ser Ile 27 O 275 280 Patent Application Publication Jan. 6, 2005 Sheet 2 of 25 US 2005/0003473 A1
gag togg titc gag Ciga gag Citg ggit CCC aac CaC gaC gC g g to Ctg aag 1036 Glu Trp Phe Glu Arg Glu Lieu Gly Pro Asn His Asp Ala Val Leu Lys 285 290 295 cag g to acg Ctc. tcc at a gtt gct atc. cac acc acg agt gac cita citc 1084 Glin Val Thr Leu Ser Ile Val Ala Ile His Thr Thr Ser Asp Leu Leu - 3OO 305 31 O ttg cag gcc atg agc gat citc gcg cag aac cc.g aaa gtg Cta caa goa 1132 Leu Gln Ala Met Ser Asp Leu Ala Glin Asn Pro Llys Val Lieu. Glin Ala 315 320 325 gtg.cg C gag gag gtg, g to Cg a gtg Ctg agc a CC gag ggg Ctic agc aag 80 Val Arg Glu Glu Val Val Arg Val Leu Ser Thr Glu Gly Leu Ser Lys 330 335 340 345 g to tcg citt cac agt citc aag Ct c atg gac agc gcg ttg aag gaa agc 1228 Val Ser Leu. His Ser Leu Lys Leu Met Asp Ser Ala Leu Lys Glu Ser 350 355 36O Cag Cgt. Ctc agg cCt acg Ctt CtC ggc ticc titt cqt cgg cag goa acg 1276 Gin Arg Leu Arg Pro Thr Leu Leu Gly Ser Phe Arg Arg Glin Ala Thr 365 370 375 aat gac atc aag ctgaag agc ggg ttt g to ata aag aaa ggg act aga 1324 Asn Asp Ile Llys Leu Lys Ser Gly Phe Wall Ile Lys Eys Gly Thr Arg 380 385 390 gtc gtg atc gac agc acc cat atg togg aat ceC gag tat tac act gac 1372 Val Val Ile Asp Ser Thr His Met Trp Asin Pro Glu Tyr Tyr Thr Asp 395 4 OO 4 OS cct citc cag tac gac ggg tac Cgc tac titc aac aag cgg cag aca ccc 420 Pro Leu Glin Tyr Asp Gly Tyr Arg Tyr Phe Asn Lys Arg Glin Thr Pro 40 415 420 425 ggc gag gac aag aac gCg ttg Ctic gtc agc aca agc gcc aac cac atg 1468 Gly Glu Asp Lys Asn Ala Leu Lieu Val Ser Thr Ser Ala Asn His Met 430 435 440 gga titc ggt cac ggc gtt CaC gCC tigt CCt ggC aga titc ttC goc toc 156 Gly Phe Gly. His Gly Val His Ala Cys Pro Gly Arg Phe Phe Ala Ser 445 450 455 aac gag atc aag att goc ttg tt Cat at C atc tta aat tat gag tgg 1554 Asn Glu Ile Lys Ile Ala Leu Cys His Ile I e Leu Asn Tyr Glu Trip A 60 465 470 cgt. Ctt cca gac ggc titC aag CCC Cag CCt. Ctc aac atc ggg atg act 612 Arg Leu Pro Asp Gly Phe Lys Pro Gln Pro Leu Asn Ile Gly Met Thr A 75 480 485 tat Ctg gcg gat CCC aat acc agg atg Ctg atc agg CCa cqC aag gog 1660 Tyr Lieu Ala Asp Pro Asn Thr Arg Met Leu Ile Arg Pro Arg Lys Ala A 90 495 500 505 gag atc gat at g g cg agt tta act g td tag g togaacacg aagtoctgat 1710 Gu Ile Asp Met Ala Ser Leu Thr Val * 510 gaagtgttat tigg to agtgg gtgaagcaag, tcgcagaaat gtgtaacaat ttataagaat 1770 aaaa. 1776 Patent Application Publication Jan. 6, 2005 Sheet 3 of 25 US 2005/0003473 A1 Figure 2 - Nucleotide and protein sequence of human ocidoreductase
atg gga gac toc cac gitg gac acc agc ticc acc gtg tCC gag gCg gtg 48 Met Gly Asp Ser His Val Asp Thr Ser Ser Thr Val Ser Glu Ala Val . 1. 5 10 15 gcc gaa gaa gta tot Ctt ttc agc atg acg gac atg att ctg. t t t tog 96 Ala Glu Glu Val Ser Leu Phe Ser Met Thr Asp Met Ile Leu Phe Ser 20 25 30 Ctc atC gtg ggt Citc Cta acc tac togg t t c ct c ttic aga aag aaa aaa 144 Leu Ile Val Gly Leu Leu Thr Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45 gaa gaa gtc. ccc gag titc acc aaa att cag aca ttg acc toc tot g to 192 Glu Glu Wai Pro Glu Phe Thr Lys Ile Glin Thr Leu Thr Ser Ser Val 50 55 60 aga gag agc agc titt gtg gaa aag atg aag aaa acg ggg agg aac atc. 240 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn Tle 65 70 75 80 atc gtg ttc taC ggC to C cag acg ggg act gca gag gag titt gcc aac 288 Ile Val Phe Tyr Gly Ser Glin Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 cgc ctg. tcc aag gac gcc cac cgc tac g g g atg Cga ggc atg to a gog 336 Arg Leu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 05 110 gac cct gag gag tat gac ctg gcc gac ctg agc agc ctg. cca gag atc 384 Asp Pro Glu Glu Tyr Asp Leu Ala Asp Lell Ser Ser Leu Pro Glu Ile 15 120 125 gac aac goc Citg gtg gtt titc tgc atg gCC acc tacggit gag gga gac 432 Asp Asn Ala Lieu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 140 ccc acc gac aat goc cag gac titc tac gac togg ctg cag gag aca gac 480 Pro Thr Asp Asn Ala Glin Asp Phe Tyr Asp Trp Leu Glin Glu Thr Asp - 1.45 150 155 160 gtg gat Ctc. tct ggg gtC aag titC gcg gtg ttt ggt. Ctt ggg aac aag 528 Val Asp Leu Ser Gly Val Lys Phe Ala Val Phe Gly Leu Gly Asn Lys 1.65 170 175 acc tac gag cac titc aat gcc atg ggC aag tac gitg gac aag cqg Ctg 576 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Leu 180 185 190 gag cag ct C ggC gCC Cag cqC at C titt gag Citg ggg ttg ggc gac gac 624 Glu Gln Leu Gly Ala Gin Arg Ile Phe Glu Leu Gly Leu Gly Asp Asp 195 200 205 gat gigg aac tig gag gag gac titc atc. a CC togg Cqa gag cag titC togg 672 Asp Gly Asn Leu Glu Glu Asp Phe Ile Thr Trp Arg Glu Glin Phe Trp 210 215 220 ccg gcc gtg tdt gala cac titt ggg gtg gala gCC act ggC gag gag toc 720 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240 agc att cqC Cag tac gag Ctt gtg gtc. cac acc gaC at a gat gog goc 768 Ser Ile Arg Glin Tyr Glu Lieu Val Val His Thr Asp Ile Asp Ala Ala 245 250 255 aag gtg tac at g g g g gag at g g g c cqg Ctg aag agc tac gag aac Cag 86 Lys Val Tyr Met Gly Glu Met Gly Arg Lieu Lys Ser Tyr Glu Asn Gln 260 265 270 aag CCC cc C titt gait gcc aag aat CC9 tt C Ctg gCt gca gtc. acc acc 864 Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr 275 280 285 aac cgg aag Ctg aac Cag gga acc gag cqC cac cto atg cac ctg gaa 912 Asn Arg Lys Lieu. Asn Glin Gly Thr Glu Arg His Leu Met His Leu Glu 290 295 300 ttg gaC at C tog gac toc aaa atc agg tat gaa tict ggg gac cac gtg 960 Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val
Patent Application Publication Jan. 6, 2005 Sheet 5 of 25 US 2005/0003473 A1
SO3196-00-US cag gcg g to gaC tac at C aag aaa citg atg acc aag ggc cgc tac toc 2O6 Glin Ala Val Asp Tyr Ile Lys Lys Leu Met Thr Lys Gly Arg Tyr Ser 660 665 670 ctg gaC gtg tgg agc 2031. Leu Asp Val Trp Ser 675
Patent Application Publication Jan. 6, 2005 Sheet 7 of 25 US 2005/0003473 A1
ttic aac gtc. aag gac cgc aac togt ctg. cac atg gala atc agc atc gcc. 1150 Phe Asn Val Lys Asp Arg Asn Cys Lieu. His Met Glu Ile Ser Ile Ala 295 300 305 gg t agc aac citc act tac cag act ggit gac cac atc gct gtt togg ccc 1198 Gly Ser Asn Leu Thr Tyr Glin Thr Gly Asp His Ile Ala Val Trp Pro 310 . 31.5 320 acc aac goc ggt toc gag g to gat cgg ttc ctg cag gCt titt gg. t c to . 1246 Thr Asn Ala Gly Ser Glu Val Asp Arg Phe Leu Glin Ala Phe Gly Leu 325 330 335 gaa gga aag cqc cac toc g to atc. aac att aag gg t atc gat gtg acc 1294 Glu Gly Lys Arg His Ser Val Ile Asn ille Lys Gly Ile Asp Val Thr 340 345 350 gct aag gtt cog at t coc act cot acg acc tat gaC gcc gca gtt cgc 1342 Ala Lys Val Pro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala Val Arg 355 360 365 370 tac tac Citg gaa gtc. togt gCC CCC gtt toc cgt cag titt g to tcg act 1390 Tyr Tyr Lieu Glu Val Cys Ala Pro Val Ser Arg Glin Phe Val Ser Thr 375 380 385 ctic gct gcc titt gcc cot gat gaa gog acc aag gCg gag atc gtt cgt 1438 Leu Ala Ala Phe Ala Pro Asp Glu Ala Thr Lys Ala Glu Ile Val Arg 390 395 400 ttg ggit ggc gac aag gac tat ttic cat gag aag att acc aac cqa togc 1486 Leu Gly Gly Asp Lys Asp Tyr Phe His Glu Lys Ile Thr Asn Arg Cys 405 410 415 ttc aac atc got cag gCt c to cag agc atc acg toc aag cot t to acc 1534 Phe ASn Ile Ala Glin Ala Leu Glin Ser Ile Thr Ser Lys Pro Phe Thr 420 425 430 gcc gtc. cc.g. t t c toc ctg. Ctt atc gaa ggit atc acc aag citt cag ccc 1582 Ala Val Pro Phe Ser Leu Leu Ilie Glu Gly Ile Thr Lys Leu Glin Pro 435 440 445 450 cgit tac tact cq atc. tcc tog tot toc ctg gtt cag aag gac aag att 1630 Arg Tyr Tyr Ser Ilie Ser Ser Ser Ser Leu Val Glin Lys Asp Lys Ile 455 460 465 agc att acc gcc gtt gtg gag tog gtt cgc titg cct ggt gag gaa cac 1678 Ser Ile Thr Ala Val Val Glu Ser Val Arg Leu Pro Gly Glu Glu His 470 475 480 att g to aag ggt gtg acc acg aac tat citt cto gog citc. aag gaa aag 1726 Ile Val Lys Gly Val Thr Thr Asn Tyr Lieu. Leu Ala Leu Lys Glu Lys 485 490 495 cala aac ggc gag cct tcc cct gaC ccg. ca C ggc titg act tac tot atc 1774 Gln Asn Gly Glu Pro Ser Pro Asp Pro His Gly Leu Thr Tyr Ser Ile 500 SOS 510 act gga ccc cgt. aac aag tac gat ggc atc cat gtC ccc gtt cac gtc 1822 Thr Gilly Pro Arg Asn Lys Tyr Asp Gly Ile His Val Pro Val His Val 55 520 S25 530 cgc cac tog aac t t c aaa ttg ccc. tcg gat coc tog cga cct gtg atc 1870 Arg His Ser Asn Phe Lys Leu Pro Ser Asp Pro Ser Arg Pro Vali Ile 535 S40 545 atg gtt gga CCC ggit act ggit gtt gct cot t t c cgt ggg titt atc cag 1918 Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Arg Gly Phe Ilie Glin 550 555 560 gag cqt gct gCC titg gCC gCg aag ggC gag aag g to gga act acc ttg 1966 Glu Arg Ala Ala Leu Ala Ala Lys Gly Glu Lys Val Gly Thr Thr Leu 565 570 575 citt ttc ttC ggc tgc cgt aag toc gac gaa gat ttc ttg tac aag gat 2014 Leu Phe Phe Gly CyS Arg Lys Ser Asp Glu Asp Phe Leu Tyr Lys Asp 58O 585 590 gaa tigg aag act ttt Cag gag cag ct t ggc gac tog ctic aag atc atc 2062 Glu Trp Lys Thr Phe Glin Glu Gln Leu Gly Asp Ser Leu Lys Ile Ile 595 600 605 610 act gcc titc. tct C9t gaa tog gct gag aaa gtC tac g to cag cac agg 2110 Thr Ala Phe Ser Arg Glu Ser Ala Glu Lys Val Tyr Val Glin His Arg 615 620 625 Ctg cgt gag Cat gCC gag Citg g to agt gac Citg Ctg aag cag aaa gcc 21.58 Patent Application Publication Jan. 6, 2005 Sheet 8 of 25 US 2005/0003473 A1
Leu Arg Gu His Ala Glu Lieu Val Ser Asp Leu Lleu Lys Gln Lys Ala 630 635 640 act ttc tat gtt tac ggt gac 'gct gcc aac atg gcc cgt gaa gtc aac 22O6 Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu Val Asn 645 650 655 ctic gtg citt ggg caa atc att goc aag cag cgc ggt Ct c cot gcc gag 2254 Leu Wall Leu Gly Glin Ile Ile Ala Lys Glin Arg Gly Leu Pro Ala Glu. 660 665 670 aag ggc gag gag atg gtg aag CaC atg CgC agc agc ggC agc tac Cag 23 O2 Lys Gly Glu Glu Met Val Lys His Met Arg Ser Ser Gly Ser Tyr Glin 675 680 685 690 gac gat gtC togg tcc taa aa 2322 Asp Asp Val Trp Ser * 695
Patent Application Publication Jan. 6, 2005 Sheet 10 of 25 US 2005/0003473 A1
CAA75565 283 cAB91316 274 CAB56503 259 AAB 94.588 276 pMON45624 269 CAA75566 274 AAD34552 282 CAA755 67 129 CAA76703 282 CAA57874 44 CAA9268 277
CAA755.65 338 KESQRLK CAB91316 324 LDSVKESQRivk CAB56503 314 Ek first Wr NWF Nor v VDETKLHQLKYLOAVKELRL pMON45624AAB94588 321329 Strida VWar WDETE DSAKESORLRSEE - CAA75566 332 GRHPEYIEPLREVVOREEG------EDSA y AAD34 552 336 : ''W'. ... - - - - - CAA75567 186, CAA76703 333 LDSFKESQRFN
CAA57874 96 DRFNSPDLSMSNL CAA91268 336 LRFY-P------
CAA755.65 384 CAB936 370 CAB56503 36 PSKSRIV AAB94588 376 pMON45624 367 G E. CAA75566 378 GF ESSEN AAD3 4 552 382 CAA75567 238 - SEETpTFSpirS CAA76703 379 AMLQ------DSAHWPG
CAA57874 152 KNYKLCESLTGHS HK------DHKL CAA91268 382 ------PHFS VCLPETRHR------NPEN
CAA75565 427 PLK CAB91316 413 AEK SEE,RETPGAGKENVAQL E3EEE PEH CAB56503 404 PEK- - - - - m - - - m - - - - - VPFKSNSFFYPFG AAB94588 419 TES pMON45624 410 CAA75566 421 AAD34552 4.25 CAA75567 289 CAA76703 423 CAA57874. 205 CAA91268 425
CAA75 S65 48O SHLKYPEKPV----- EGSSMEPRK EE S -EEAI - - - - - CAB91316 468 EE - - - - - EGSDPKIRTFGFSMGVDPSLRVEYKGRQ-PEEL----- CAB56503 449 L PWNFS-SSSPA- - - - - AAB94588 464 RRQNDLCLIP - - - P ------pMON45624 463 ALCHENYEARLP----- DGFKPQPLNI -AEDMASLTW CAA75566 474 ALCHLiybwkLC----- PDTETKPDT ES5 DILIRRRESVEEDLEAI - - AAD34552 478 MLAYLLERYDWKV- - - - - PDEPLQYYRHSFSVRIHPTTSLMMRRRD-EDRLPGSL CAA75567 347 LE----DGKPGPELMRVGTETRLDTKAGLERRR------CAA76703CAA57874. 258479 YDIKLP-- E. - - - - -DGLSRPKNIEFEWLASLNACANA------LCVRKRS---RDE---- CAA9268 468 ELKOF----EGEADLIPDCNGRIMRPNDPVRLHEKPRN------CAA75565 (SEQ ID NO: 27) CAB91316 (SEQ ID NO: 28) CAB56503 (SEQ ID NO: 29) AAB94588 (SEQ ID NO: 30) pMON45624 (SEQ ID NO: O2) CAA75566 (SEQ ID NO: 31) AAD34552 (SEQ ID NO: 32) CAA75567 (SEQ ID NO: 33) CAA76703 (SEQ ID NO: 34) CAA57874 (SEQ ID NO: 35) CAA91268 (SEQ ID NO: 36) Patent Application Publication Jan. 6, 2005 Sheet 11 of 25 US 2005/0003473 A1 Figure 5 - Phylogenetic tree showing the relatedness of Aspergillus ochraceus 11 alpha hydroxylase to the top 10 BLAST hits from GenBank
pMON45624 CAA75566 AAD 54-552 CAA/6/O 5 CAA75567 CAA57874 CAA9 1268 CAB565O3 - AAB94588 CAAA 55.65 CAB9 1316 Patent Application Publication Jan. 6, 2005 Sheet 12 of 25 US 2005/0003473 A1 Figure 6 - Percent homology of Aspergillus ochraceus 11 alpha hydroxylase to the top 10 BLAST hits from GenBank
Accession Number Species % D to 11a. OH CAB91316 Neurospora crassa 40
CAA76565 - Gibberella fujikuroi 37 CAA7 5566 Gibberella fujikuroi 37
AAD34552 Aspergillus terreus) 29
CAA75567 Gibberella fujikuroi 24
CAA57874 Fusarium oxysporum 24 CAA76703 Gibberella fujikuroi 23
CAB56503 Catharanthus roseus 14
AAB94588 Glycine max 14
CAA91268 Caenorhabditis elegans 12 Patent Application Publication Jan. 6, 2005 Sheet 13 of 25 US 2005/0003473 A1 Figure 7 - Amino acid homology alignment of A. ochraceus and human oxidoreductase to NADPH cytochrome P450 reductases from A. niger, mouse, and S. cerevisiae PMON45 605 . TDALFSLIVGLTYWFLF SEE humanIOSe sa LFSLIVGELTYWFLFBKKKEEPEFAKIOTESEivity fire:Eg pMON45632 s niger yeast
PMON45 605 P-AHRGMRGMSADPEEYDL hunan OSes EEEEEE pMON45632 niger yeast
PMON45 605 i20 LVVFCMATYGEGDPTDNAQDFYDLO human 120 LVFCMATYGEGDPTDNAQDFYDELQ------ET----- rose 120 ADLSSLPEIDKSLVVFCMATYGEGDPTDNAQDFYDLQ------ET----- pMON45632 107 ES NE FYIQETGEDAAFESGATP yeastniger 107O2 E. -AFFVATYGEGEPTDNAVEFyoffrcDDVAFES-ASADE. vSFE5CN
PMON45 605 169 human 69 OlSe 169 pMON45632 1.65 niger 164 yeast 148
humanPMON45 605 228 CEHFGEATGEESSIROYEEEEEEEE D- - - -D Olse 228 CEFR, ATGEESS O pMON45632 225 SEAMNE YEPVFNVTEDESLSP niger 224 SESMDEE---EREAYEPVFCVTENESLSP yeast 208 KELHE---EEAKFTSFQYTVLN---EITDSSLGEPSAHYLPSHQLNRNADGICL
PMON45605 275 A. 3. IRYESGDHVAVYPANDSALVNQL human 275 AA B EEEEEENS: pMON45632OSe 275273 : EVEN iger 272 yeast 262
PMON45 605 335 human 335 rose 335 pMON45632 333 niger 332 yeast 322
PMON45605 393 A humanClSe 393 S-EPSEQEHLKMAsssESESS EGKELYLSWWEE pMON45632 391 niger 390 MRSR yeast 378 PNAD
PMON45 605 451 human 451 Inouse 45 pMON45632 448 niger 4A 6 yeast 436 Patent Application Publication Jan. 6, 2005 Sheet 14 of 25 US 2005/0003473 A1
PMON45 605 503 ------human 503 ------NGGR IOSe 503 ------NG pMON45632 505 PDPHG-ETYSITGPRNKS niger 503 E. O SN S yeast 495 AETNLP r is FRLPSNP
PMON45 605 - - -WGEL ELAQFHRD GALTOLNWAFSRE human ---VGETLLYGCRRSDEDSLYREELAQFHRD-GALTQLNVAFSRE Olli Se - - -WGETLL scRRS5E5SEREE FHKD-GALTQLNWAFSRE pMON45632 EK---VGTTLLFFGCRKSDEDELYREWKT SRE niger EK- - -WGP SE yeast NNVSGKHL N
PMON45 605 human pMON45632ITOl:Se Evsplit-KQKATFYvcGD niger DKQKATFYVCGDA yeast
PMON45 605 human OlSe pMON45632 niger yeast PMON45 605 (SEQ ID NO: O3) human (SEQ ID NO: O6) IOSe (SEQ ID NO: 39) PMON45632 (SEQ ID NO : 05) niger (SEQ ID NO: 38) yeast (SEQ ID NO : 37) Patent Application Publication Jan. 6, 2005 Sheet 15 of 25 US 2005/0003473 A1 Figure 8 - Amino acid homology alignment of A. ochraceus oxidoreductase to NADPH cytochrome P450 reductases from A. niger and S. cerevisiae A. niger A. ochraceus S. cerevisiae A. niger 60 SETGKNCVIFYGSOTGTAEDYASRLAKEGSORFGLKTMVADLERYDYENLC OFPEDKVAF A. ochraceus 60 BETGKNCVIFYGSOTGTAEDYASRLAKEGSORFGLKTMVADLEYDYENLE FPEDKVVF S. cerevisiae 55 iCADENYDESL VS A. niger 120 AFGLGNNTYEHYNA. A. ochraceus 120 FGLGNNTYEHYNA. S. cerevisiae 113 A. niger 79 A. ochraceus 80 S. cerevisiae 163 A. niger 239 A. ochraceus 240 GEPKGPYSANPFIAPIEEEEEEEEE S. cerevisiae 223 SHQLNRNADGIOLGPFDLSGPSIAPI A. niger 290 A. ochraceus 291 S. cerevisiae 280 A. niger 350 A. ochraceuS 35 S. cerevisiae 338 A. niger 4.09 A. ochraceuS 411 VPFSLLEGITKLQPRYYSISSSSLVQKDKISIT S. cerevisiae 398 ES A. niger 468 A SWRLP--GASHMVKGVTTNYLLALKOKONGRSSRPSR-LDLLHHGPRNKYDGIHV A. ochraceus 470 AVWESVRLP--GEEHiWKGWTTNYLLALKEKQNGEPSPDPHG S. cerevisiae 458 SiveNFPNPELPEAPPGVGvTNLRNOLAONNVNIAETNLPHyd A. niger 525 A. ochraceus 527 S. cerevisiae 518 A. niger 579 FGCRKSDEDFLYKDEWKTYQQLGDNLKIITAFSRE-GPQKVYVQHRLREHSELVSDLLK A. ochraceus 581 FGCRKSDEDFLYKDEWKTEEL S. cerevisiae 578 A. niger 638 OKATFYVCGDAANMAREVNLVLGQIIAA A. ochraceus 640 S. cerevisiae 637 NGAFIYvogDSEESSE GISR A. ochraceus, PMON45632 (SEQ ID NO: 05) A. niger (SEQ ID NO: 38) S. cerevisiae, yeast (SEQ ID NO: 37) Patent Application Publication Jan. 6, 2005 Sheet 16 of 25 US 2005/0003473 A1 Figure 9 - Phylogenetic tree showing the relatedness of Aspergillus ochraceus and human oxidoreductase to reductases from A. niger, yeast, and mouse. PMON45605 hum On pMON45632 niger yed st Patent Application Publication Jan. 6, 2005 Sheet 17 of 25 US 2005/0003473 A1 Figure 10 - Percent homology between Aspergillus ochraceus oxidoreductase to reductases from A. niger, yeast, and mouse and human.
Accession number organism % id to Aoch oxred
CAA8550 A. niger 84
BAA02936 S. cereuisiae 37
BAA04496 OSe 34
AAB21814 human 33
Patent Application Publication Jan. 6, 2005 Sheet 19 of 25 US 2005/0003473 A1
Olse 360 PFPCPTYRTALTYYLDITNPPRTNVLYELAQYASEPSEQEHLEKMASSSGEGKELYLSW pig 360 PFPCPT RTALTYYLDITNPPRTNVLYELAQYASEPSEOEQLRKMASSSGEGKELYLS,
PMON45 605 420 ARRELAILODCPSLRPIPELLOELAPRILOARYSIASSSKVHPNSWHICAVVVEYE human 420 rabbit 421 VVEARRHILAILODYPSLRPPIDHL,CELLPRLQARYYSIASSSKVHPNSWHICAVAVEYE rat 420 VVEARRHILAILQDYPSLRPPIDHECELLPRLOARYYSIASSSKVHPNSWHICAVAVEY OS 420 VVEARRHILAILODYPSLRPPIDHLCELLPRLOARYYSIASSSKVHPNSWHICAVAVEY pig 420 ARRHILAILODYPSLRPPIDHLCERLPRLOARYYSIASSSKVHPNSWHICAVWWEYE
PMON45 605 480 TKAGRNKGVATNWLRAKEPAGENGGRALVPMFVRKSOFRLPFKATTPWIMWGPGTGWAP human 480 TKAGRsNKGVATNLRAKEPAGENGGRALVPMFVRKSOFRLPFKATTPVIMVGPGTGVAP rabbit 481 TKAGRNKGVATSNLRAKEPAGENGGRALWPMFVRKSOFRLPF KATTPWIMWGPGTGWAP Eat 480 AKSGRVNKGVATSWLRAKEPAGENGGRALWPMFvRKSOFRLPFKSTTPvMVGPGTGIA Ol See 480 AKSGRVNKGVATswLR KEPAGENGRRALWPMFVRKSOFREPFKPTTPVIMVGPGTGWAP pig A 80 TKSGRVNKGwaTswlRAKEPAGENGRRALvPMFvRKSOFRLPFKATTPvimvcPGTGwap Patent Application Publication Jan. 6, 2005 Sheet 20 of 25 US 2005/0003473 A1
PMON45 605 540 human 540 FIGFIOERAWERQOGKEvGETLLYYGCRRSDEDYLYREELAOFHRDGALTOLNWAFSREO rabbit 541 FIGFIOERAWLROOGKEvGETLLYYGCRRAAEDYLYREELAGEOKDGILSQLNWAFSREC rat 540 FMGFIOERAWLREOGKEvGETLLYYGCRRSDEDYLYREELARFHKDGALTOLNvAFSREC OS6 540 FMGFIOERAWLREOGKEvGETLLYGCRRSDEDYLYREELARFHKDGALTOLNWAFSREC pig 540 FireFICERAWLQEOGKEvGETLLYYGCRRSDEDYLYREELAOFHAKGALTRLSWAFSREC
PMON45605 600 SHKviYvoHLLKoDREHLWKL-EGGAEYvcGDARNMARDVONTFYDIVAELGAMEHAOa. human 600 SHKWYWOHLLKODREHLWKLI-EGGAHIYWCGDARNMARDVONTFYDIVAELGAMEHAOZ
rabbit
rat w OSe 600 AHKVYWOHLLKRDKEHLWKLIHEGGAHIYvcGDARNMAKDvoNTFYDIVAEFGPMEHTO pig 600 POKVyvOHLLKRDKEHLWKLIHDGGAHIYCGDARNMARDvoNTFCDIVAEoGPMEHAoi
PMON45605 659 VDYKKLMTKGRYSLDws (SEQ ID NO: 03) human 659 (SEQ ID NO: 52) rabbit 661 (SEO ID NO : 53 ) rat 660 (SEQ ID NO: 54 ) IOUS 62 660 VDYWKKLMTKGRYSLDVWS (SEQ ID NO: 55) pig 660 VDYWKKLMTKGRYSLDVWS (SEQ ID NO: 56) Patent Application Publication Jan. 6, 2005 Sheet 21 of 25 US 2005/0003473 A1 Figure 12- Phylogenetic tree showing the relatedness of human oacidoreductase (P16435) with top 4 hits from SuissProt PMON45605 humon pig rCit
O US 6 robbit Patent Application Publication Jan. 6, 2005 Sheet 22 of 25 US 2005/0003473 A1 Figure 13- Percent homology between human oxidoreductase and top 4 hits from Swiss Prot
Accession number Species %id to human oxred
P00388. rat 92 poss rabbit 92
P37040 OS 92
P04175 pig 91 Patent Application Publication Jan. 6, 2005 Sheet 23 of 25 US 2005/0003473 A1 Figure 14 - Expression of Aspergillus ochraceus 11 alpha hydroxylase in transfected Sf9 insect cells Expression of 11-o-Hydroxylase in Transfected Sf9 Cells
Patent Application Publication Jan. 6, 2005 Sheet 24 of 25 US 2005/0003473 A1 Figure 15- Expression of Aspergillus ochraceus P450 oacidoreductase in transfected Sf9 insect cells Expression of Fungal P-450 Oxidoreductase in Transfected Sf9 Cells
OO V. CN Patent Application Publication Jan. 6, 2005 Sheet 25 of 25 US 2005/0003473 A1 Figure 16 - Conversion of androstenedione to 11 alpha hydroary androstenedione monitored by HPLC
60 andones 40 3. E 2O 110-hydroxyandrostenedione
40 s S 2O C
O O 5 1O 15 20 25 HPLC Retention Time (min.) US 2005/0003473 A1 Jan. 6, 2005
ASPERGILLUS OCHRACEUS 11 ALPHA 0005 Bioconversion reactions have been widely used in HYDROXYLASE AND OXDOREDUCTASE the field of steroids (Kieslich, K.; Sebek, O. K. Annu. Rep. Ferment. Processes 3:275-304, 1979; Kieslich, Klaus. Econ. PRIORITY Microbiol., 5 (Microb. Enzymes Bioconvers.): 369-465, 1980). A variety of reactions have been characterized, 0001. The present application claims priority under Title including hydroxylation, epoxidation, oxidation, dehydro 35, United States Code, S 119 of U.S. Provisional Applica genation, ring and Side chain degradation, reduction, tion Ser. No. 60/244,300, filed Oct. 30, 2000. hydrolysis, and isomerization reactions. Many types of microorganisms have also been used including Species as FIELD OF THE INVENTION diverse, for example, as Acremonium, Aspergillus, Rhizopus, 0002 The present invention relates to a novel cyto Fusarium, Penicillium, Streptomyces, Actinomyces, Nocar chrome P450-like enzyme (Aspergillus Ochraceus 11 alpha dia, Pseudomonas, Mycobacterium, Arthrobacter and Bacil hydroxylase) and an oxidoreductase (Aspergillus OchraceuS lus. oxidoreductase) isolated from cDNA library generated from 0006 A variety of approaches have been used to facilitate the mRNA of Aspergillus Ochraceus spores. When the the hydroxylation of intermediates used in the Synthesis of cDNA encoding the 11 alpha hydroxylase was co-expressed commercially-important steroid compounds. U.S. Pat. No. in Spodoptera frugiperda (Sf-9) insect cells with the cDNA 4,588,683, for example, describes a method of preparing 11 encoding human oxidoreductase as an electron donor, it beta, 17 alpha, 20, 21 tetrahydroxy steroids by incubating Successfully catalyzed the conversion of the Steroid Substrate Substrate compounds in a medium comprising a fungal 4-androstene-3,17-dione (AD) to 11 alpha-hydroxy-AD as culture of the genus Curvularia capable of effecting 11 beta determined by HPLC analysis. The invention also relates to hydroxylation. Aspergillus OchraceuS cultures and prepara nucleic acid molecules associated with or derived from these tions of mycelia have also been used to convert progesterone cDNAS including complements, homologues and fragments and other Steroids to their corresponding 11 alpha hydroxy thereof, and methods of using these nucleic acid molecules, forms (Tan, L. and Falardeau, P., 1970; Tan L., and to generate, for example, polypeptides and fragments Falardeau. P. J. Steroid Biochem. 1:221-227, 1970; Samanta, thereof. The invention also relates to the generation of T. B. et al., Biochem. J. 176,593-594, 1978; Jayanthi, C. R. antibodies that recognize the A. OchraceuS 11 alpha et al., Biochem. BiophyS. Res. Commun. 106:1262–1268, hydroxylase and oxidoreductase and methods of using these 1982). antibodies to detect the presence of these native and recom 0007. The advent of new and expanded clinical uses of binant polypeptides within unmodified and transformed host steroids for the treatment of a wide variety of disorders has cells, respectively. The invention also provides methods of created a need for improved methods for the production of expressing the Aspergillus 11 alpha hydroxylase gene Sepa Steroid compounds and their intermediates on a commercial rately, or in combination with human or Aspergillus oxi scale. U.S. Pat. No. 4,559,332, for example, describes a doreductase, in heterologous host cells, to facilitate the number of methods for the preparation of 20-Spiroxane bioconversion of steroid substrates to their 11 alpha Series of Steroid compounds, including methods for the hydroxy-counterparts. preparation of eplerenone methyl hydrogen 9,11C.-epoxy 17O-hydroxy-3-oxopregn-4-ene-7C,21-dicarboxylate, Y-lac BACKGROUND OF THE INVENTION tone (also referred to as eplerenone or epoxymeXrenone) and 0.003 Microbial transformation or bioconversion reac related compounds. WO 98/25948 and U.S. application Ser. tions have long been used to facilitate the chemical Synthesis No. 09/319,673 describe novel processes for the preparation of a wide variety of pharmaceutical products. Stereospecific of 9,11-epoxy Steroid compounds, especially those of the reactions carried out under mild enzymatic conditions fre 20-Spiroxane Series and their analogs, novel intermediates quently offer advantages over comparable chemical pro useful in the preparation of Steroid compounds, and pro ceSSes which result in undesireable Side products. Microor ceSSes for the preparation of Such novel intermediates. U.S. ganisms also have the ability to carry out Simultaneous Pat. No. 6,046,023 discloses improved methods for the independent or Sequential reactions on a Substrate molecule, microbial transformation of canrenone or estr-4-ene-3,17 minimizing the number of distinct Steps in a Synthesis and dione into its 11 O-hydroxy analogue using microorganisms reducing the total cost of the desired intermediate or end of the genus Aspergillus, Rhizopus, and PeStelotia, using product. steroid substrates having a purity of less than 97% and more than 90% at a concentration greater than 10 g/L. 0004 General features of microbial systems used as biocatalysts for the transformation of organic compounds 0008 Many modern, systematic approaches needed to has been reviewed (See e.g., Goodhue, Charles T, Microb. optimize bioconversion of particular Steroid intermediates Transform. Bioact. Compa, 1:9-44, 1982). Biotransforma are often hindered by insufficient biochemical knowledge of tions can be carried out, for example, in continuous cultures the enzymes involved in their Synthesis and degradation. or in batch cultures. Enzymes Secreted from the microor Eukaryotic cytochromes P450 appear to be associated with ganism react with a Substrate, and the product can be the endoplasmic reticulum (ER) or mitochondrial mem recovered from the medium. Intracellular enzymes can also branes. The electron donor for ER-associated cytochrome react with a substrate if it is able to enter cells by an active P450 enzymes is often an FAD/FMN-dependent NADPH or a passive diffusion proceSS. Immobilized, dried, perme cytochrome P450 oxidoreductase. Electron transfer in the abilized, and resting cells, and Spores have also been used mitochondrial cytochromes P450 is usually mediated by an for microbial transformations. The use of cell extracts and NADPH-ferredoxin oxidoreductase and ferrodoxin. The purified enzymes in Solution, or immobilized on carriers, Specific electron donors known to be involved in mamma may eventually offer Significant cost or control advantages lian Steroidogenesis, are also called adrenodoxin reductase over traditional fermentation methods. and adrenodoxin, respectively. US 2005/0003473 A1 Jan. 6, 2005
0009 While fungal biotransformations are known to be catalyzing an oxidation Step involved conversion of choles mediated by cytochrome P450 enzymes, many of these terol into hydrocortisone, including the conversion of cho enzymes are extremely difficult to purify in an enzymati lesterol to pregnenolone; the conversion of pregnenolone to cally-active form (van den Brink et al., Fungal Genetics and progesterone; the conversion of progesterone to 17 O.-hy Biology 23, 1-17, 1998). Many fungal P450 enzymes appear droxy-progesterone; the conversion of 17O-hydroxyproges to be associated with the endoplasmic reticulum (van den terone to cortexolone; and the conversion of cortexolone to Brink et al., Fungal Genetics and Biology 23, 1-17, 1998). hydrocortisone. Yeast have an adrenodoxin reductase homologue which was shown to couple with a mammalian 11 beta hydroxylase in 0014. The sequences of Aspergillus ochraceus 11 alpha vitro. (Lacour et al., Journal of Biological Chemistry 273, hydroxylase and A. Ochraceus oxidoreductase have not been 23984-23992, 1998). In contrast, the electron donor which reported. Knowledge about their Sequence could greatly couples with Aspergillus OchraceuS 11 alpha hydroxylase facilitate the development of expression vectors and recom binant host Strains that can carry out more efficient biocon was predicted to be an NADPH-cytochrome P450 oxi versions of Steroid intermediates and the Synthesis of end doreductase (Samanta and Ghosh, J Steroid Biochem 28, products on a commercial Scale without the problems asso 327-32, 1987). The steroid 11 alpha hydroxylation complex ciated with partially-characterized host Strains or an incom in Rhizopus nigricans also appears to require an NADPH plete understanding of the enzymes involved in Steroido cytochrome p450 oxidoreductase (Makovec and Breskvar, genesis. The present invention overcomes many of the Arch Biochem Biophys. 357, 310-6, 1998). Amplification of limitations discussed above by identifying enzymes capable cytochrome R. nigricans P450 and NADPH-cytochrome of carrying out the 11 alpha hydroxylation of Steroids. This P450 reductase activities in preparations of progesterone approach not only greatly facilitates the use of 11 alpha induced fungal mycelia may the facilitate biochemical char hydroxylation, but also permits the development of new acterization of both enzymes (Makovec and Breskvar, Strategies for the identification of Similar enzymes from Pflugers Arch-Eur J. Physiol 439(Suppl). R111-R112, other fungi, the cloning of other enzymes involved in 2000). Steroidogenesis from Aspergillus OchraceuS and other 0.010 Aspergillus Ochraceus spores have been shown to microorganisms, and the development of improved host catalyze the 11 alpha hydroxylation of Steroid Substrates Strains or methods using free cells or immobilized cells or such as progesterone (Dutta T K, Datta J, Samanta T B, enzymes in bioconversion reactions. Similar approaches Biochem. Biophys. Res. Commun. 192:119-123, 1993). A. could also be developed to aid in the construction of fumigatus is also known to exhibit a steroid 11 alpha expression vectors and recombinant host strains that are hydroxylase activity (Smith et al., J Steroid Biochem Mol more amenable to propagation and control than wild-type Biol 49:93-100, 1994). The A. fumigatus enzyme is distin microorganisms now commonly used for bioconversion in guished from the A. Ochraceus enzyme, in that it appears to large Scale bioreactors. be a cytochrome P450 with dual site-specificity for 11 alpha and 15 beta hydroxylation and, unlike the A. Ochraceus SUMMARY OF THE INVENTION hydroxylase, appears to be non-inducible. 0015. In its broadest scope, the present invention pro 0.011 Despite recent advances in sequencing technolo vides a method to clone enzymes involved in Steroid gies, detailed knowledge about the Structural relationships of metabolism and use of these enzymes to produce novel fungal cytochrome P450s gleaned from nucleotide Sequence Steroid intermediates and end-products. One aspect of the data remains primitive. Breskvar et al., (Biochem. BiophyS. claimed invention is to provide a novel enzyme 11 alpha Res. Commun 1991; 178, 1078-1083, 1991) have described hydroxylase and oxidoreductase, and their nucleic acids, a genomic DNA sequence from Rhizopus nigricans for a proteins, peptides, fragments, and homologues. The inven putative P-450 encoding an 11O-hydroxylase for progester tion also relates to methods of identifying and cloning other one. This Sequence may not be complete, however, Since the enzymes involved in steroid metabolism. The invention also predicted amino acid Sequence lacks the canonical heme covers novel vectors and host cells, a novel method for binding motif, FXXGXXXCXG, which is common to almost making heterologous proteins by using the above vectors, all known cytochrome P-450 enzymes. (Nelson et al., Phar and a method for identifying the Substrate Specificity of the macogenetics 6:1-42, 1996). cloned enzymes. 0012. The cloning and characterization of the NADPH 0016. The invention provides a means for determining cytochrome P450 oxidoreductase (cprA) gene of Aspergillus the Substrate Specificity of the cloned 11 alpha hydroxylase, niger has been described (van den Brink, J., et al., Genbank allelic variants, muteins, and fusion proteins thereof, per accession numbers Z26938, CAA81550, 1993, unpub mitting evaluation of a broad array of Steroid Substrates lished). The primary structure of Saccharomyces cereuisiae including 3 keto delta 4.5 steroids (3 keto delta 4 steroids); NADPH-cytochrome P450 reductase has also been deduced 3 keto delta 4, 5 delta 6, 7 steroids (3 keto delta 4 delta 6 from the nucleotide Sequence of its cloned gene (Yabusaki et steroids); 3 keto delta 6, 7 steroids (3 keto delta 6 steroids); or 3 keto delta 1, 2 delta 4, 5 steroids (3 keto delta 1 delta al., J. Biochem. 103(6): 1004-1010, 1988). 4 Steroids). Preferred Substrates for testing include (a) can 0013 Several other approaches have been used to facili renone; (b) androstenedione; (c) aldona; (d) ADD (1,4 tate the cloning and analysis of Steroid enzymes. U.S. Pat. androstenedienedione) (e) meXrenone, (f) 6 beta meXrenone; Nos. 5,422,262, 5,679,521, and European patent EP 0528 (g) 9 alpha meXrenone; (h) 12 beta meXrenone; (ii) delta 12 906 B1, for example, describes the expression cloning of meXrenone; () testosterone; (k) progesterone; (1) meXrenone steroid 5 alpha reductase, type 2. U.S. Pat. No. 5,869,283, 6.7-bis-lactone; and (m) mexrenone 7.9-bislactone. Prefer for example, describes an expression cassette comprising ably the cloned 11 alpha hydroxylase, allelic variants, heterologous DNAS encoding two or more enzymes, each muteins, and fusion proteins thereof do not also catalyze a US 2005/0003473 A1 Jan. 6, 2005
Second hydroxylation Selected from the group consisting of canrenone; (b) androstenedione to 11 alpha hydroxy andros 15 alpha or beta hydroxylation, 6 alpha or beta hydroxyla tenedione; (c) aldona to 11 alpha hydroxyaldona; (d) ADD tion, 7 alpha or beta hydroxylation, 9 alpha or beta hydroxy (1,4 androstenedienedione) to 11 alpha hydroxy ADD; (e) lation, 12 alpha or beta hydroxylation, and 17 alpha or beta meXrenone to 11 alpha hydroxy meXrenone, (f) 6 beta hydroxylation of Substrates Selected from the group consist meXrenone to 11 alpha hydroxy 6 beta meXrenone; (g) 9 ing of 3 keto delta 4.5 steroids; 3 keto delta 4, 5 delta 6, 7 alpha meXrenone to 11 alpha hydroxy 9 alpha meXrenone; steroids; or 3 keto delta 6, 7 steroids. Most preferably the (h) 12 beta mexrenone to 11 alpha hydroxy 12 beta cloned 11 alpha hydroxylase, allelic variants, muteins, and meXrenone; (ii) delta 12 meXrenone to 11 alpha hydroxy fusion proteins thereof do not catalyze the 15 beta hydroxy delta 12 meXrenone; (ii) testosterone to 11 alpha hydroxy lation of Substrates Selected from the group consisting of 3 testosterone; (k) progesterone to 11 alpha hydroxy proges keto delta 4.5 steroids; 3 keto delta 4, 5 delta 6, 7 steroids; terone; (1) meXrenone 6,7-bis-lactone to 11 alpha hydroxy or 3 keto delta 6, 7 steroids. meXrenone 6,7-bis-lactone; and (m) meXrenone 7.9-bislac 0.017. The invention provides an isolated and purified tone to 11 alpha hydroxy mexrenone 7.9-bislactone. More nucleic acid, encoding Aspergillus OchraceuS 11 alpha preferably, the hydroxylation is Selected from the group hydroxylase. It also provides an isolated DNA, cDNA, gene, consisting of: (a) canrenone to 11 alpha hydroxy canrenone; and an allele of the gene encoding Aspergillus OchraceuS 11 (b) androstenedione to 11 alpha hydroxy androstenedione; alpha hydroxylase. Preferably the isolated and purified (c) aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4 nucleic acid is as set forth in SEQID NO: 01. Preferably the androstenedienedione) to 11 alpha hydroxy ADD. Most isolated DNA, cDNA, gene, and an allele of the gene is as preferably the hydroxylation is from canrenone to 11 alpha set forth in SEO ID NO: 01. hydroxy canrenone. 0.018. The invention provides an isolated protein having 0022. The invention also provides a method of expressing the amino acid Sequence of Aspergillus OchraceuS 11 alpha a protein that can catalyze the 11 alpha hydroxylation of 3 hydroxylase. It also provides an isolated variant of Aspergil keto delta 4.5 steroids; 3 keto delta 4, 5 delta 6,7 steroids; 3 lus OchraceuS 11 alpha hydroxylase, and a fusion protein keto delta 6, 7 steroids; or 3 keto delta 1, 2 delta 4, 5 steroids comprising this hydroxylase. Preferably the protein is as Set comprising; (a) transforming or transfecting host cells with forth in SEQ ID NO: 2. It also provides for variant of the an expression cassette comprising a promoter operably protein set forth in SEQ ID NO: 2.; a polypeptide which linked to a nucleic acid that encodes said protein, and (b) comprises SEQ ID NO: 2 with at least one conservative expressing Said protein in Said host cells. The invention also amino acid Substitution; polypeptides, with an amino acid provides for a method of producing the protein further sequence at least 99%, 95%, 90%, 75%, and 50% identical comprising the Step of recovering Said protein. Preferably, to SEO ID NO: 2. this protein is Aspergillus OchraceuS 11 alpha hydroxylase. 0019. The invention provides an isolated and purified More preferably, this method further comprises expressing nucleic acid, encoding Aspergillus OchraceuS 11 alpha OXi an electron donor protein, wherein Said electron donor doreductase. It also provides an isolated DNA, cDNA, gene, protein can donate electrons to Said protein that can catalyze and allele of the gene encoding Aspergillus OchraceuS the 11 alpha hydroxylation of 3 keto delta 4.5 steroids; 3 oxidoreductase. Preferably, the isolated and purified nucleic keto delta 4, 5 delta 6, 7 steroids; 3 keto delta 6, 7 steroids; acid, wherein Said nucleic acid Sequence is as Set forth in or 3 keto delta 1, 2 delta 4, 5 steroids. Preferably, the electron SEQ ID NO: 5. It also provides for an isolated DNA, cDNA, donor protein is Selected from the group consisting of human gene, and allele of the gene set forth in SEQ ID NO: 5. oxidoreductase and Aspergillus Ochraceus oxidoreductase. 0020. The invention provides an isolated protein having More preferably the electron donor protein is Aspergillus the amino acid Sequence of Aspergillus Ochraceus oxi Ochraceus oxidoreductase. More preferably, the nucleic acid doreductase. It also provides an isolated variant of the encoding Said Steroid 11 alpha hydroxylase and Said electron protein having the amino acid Sequence of Aspergillus donor protein are on Separate expression cassettes. More ochraceus oxidoreductase, and a fusion protein comprising preferably, the nucleic acid encoding Said Steroid 11 alpha the amino acid Sequence of Aspergillus Ochraceus oxi hydroxylase and Said electron donor protein are on the same doreductase. Preferably the isolated protein has the amino expression cassettes. Even more preferably, the Steroid 11 acid sequence set forth in SEQID NO: 6. It also provides an alpha hydroxylase is Aspergillus OchraceuS 11 alpha isolated variant of a protein set forth in SEQ ID NO: 6. a hydroxylase and Said electron donor protein is human oxi purified polypeptide, the amino acid Sequence of which doreductase. Even more preferably, the Steroid 11 alpha comprises SEQ ID NO: 6 with at least one conservative hydroxylase is Aspergillus OchraceuS 11 alpha hydroxylase amino acid Substitution; and a polypeptides with an amino and Said electron donor protein is Aspergillus OchraceuS acid sequence at least 99%, 95%, 90%, 75%, and 50% oxidoreductase. Preferably, the expression cassette is on an identical to SEO ID NO: 6. expression vector. More preferably, the expression vector is 0021. The invention provides an isolated and purified a baculovirus. Even more preferably, the baculovirus is a nucleic acid encoding an enzyme that can catalyze the 11 nuclear polyhedrosis virus is Selected from the group con alpha hydroxylation of 3 keto delta 4.5 steroids (3 keto delta Sisting of Autographa Californica nuclear polyhedrosis virus 4 steroids); 3 keto delta 4, 5 delta 6, 7 steroids (3 keto delta and Bombyx mori nuclear polyhedrosis virus. Most prefer 4 delta 6 steroids); 3 keto delta 6,7 steroids (3 keto delta 6 ably, the nuclear polyhedrosis virus is Autographa Califor steroids); or 3 keto delta 1, 2 delta 4, 5 steroids (3 keto delta nica nuclear polyhedrosis virus. Preferably, the host cells are 1 delta 4 steroids). Preferably the enzyme does not catalyze insect cells. More preferably, the insect cells are Selected the 15 beta hydroxylation of 3 keto delta 4, 5 steroids; 3 keto from the group consisting of Spodoptera frugiperda, Tri delta 4, 5 delta 6, 7 Steroids; or 3 keto delta 6, 7 steroids. choplusia ni, Autographa Californica, and Manduca Sexta More preferably, the hydroxylation is selected from the cells. Most preferably the insect cells are Spodoptera fru group consisting of: (a) canrenone to 11 alpha hydroxy giperda cells. The invention also provides a for a method of US 2005/0003473 A1 Jan. 6, 2005 expressing a protein wherein the Aspergillus OchraceuS 11 0025 The invention also provides for an expression alpha hydroxylase is SEQ ID NO: 2; the human oxidoreduc cassette comprising a promoter operably linked to an iso tase is SEQ ID NO: 4; and the Aspergillus ochraceus lated and purified nucleic acid encoding Aspergillus Ochra oxidoreductase is SEO ID NO: 6. ceus oxidoreductase. Preferably the nucleic acid is SEQ ID NO: 6. 0023 The invention also provides for an isolated and 0026. The invention also provides for an expression purified polypeptide that can catalyze the 11 alpha hydroxy cassette comprising a heterologous DNA encoding an lation of 3 keto delta 4.5 steroids (3 keto delta 4 steroids); enzyme from the metabolic pathway for the synthesis of 3 keto delta 4, 5 delta 6, 7 steroids (3 keto delta 4 delta 6 Sitosterol to eplerenone wherein Said enzyme catalyzes at steroids); 3 keto delta 6,7 steroids (3 keto delta 6 steroids); least one conversion Selected from the group consisting of: or 3 keto delta 1, 2 delta 4, 5 steroids (3 keto delta 1 delta 4 steroids). Preferably, the polypeptide does not catalyze the (a) canrenone to 11 alpha hydroxy canrenone; (b) andros 15 beta hydroxylation of 3 keto delta 4.5 steroids; 3 keto tenedione to 11 alpha hydroxy androstenedione; (c) aldona delta 4, 5 delta 6, 7 Steroids; or 3 keto delta 6, 7 steroids. to 11 alpha hydroxyaldona; (d) ADD (1,4 androstenediene More preferably, the hydroxylation is selected from the dione) to 11 alpha hydroxy ADD; (e) mexrenone to 11 alpha group consisting of: (a) canrenone to 11 alpha hydroxy hydroxy meXrenone, (f) 6 beta mexrenone to 11 alpha canrenone; (b) androstenedione to 11 alpha hydroxyandros hydroxy 6 beta meXrenone; (g) 9 alpha meXrenone to 11 tenedione; (c) aldona to 11 alpha hydroxyaldona; (d) ADD alpha hydroxy 9 alpha meXrenone; (h) 12 beta meXrenone to (1,4 androstenedienedione) to 11 alpha hydroxy ADD; (e) 11 alpha hydroxy 12 beta mexrenone; (ii) delta 12 meXrenone meXrenone to 11 alpha hydroxy meXrenone, (f) 6 beta to 11 alpha hydroxy delta 12 meXrenone; () testosterone to meXrenone to 11 alpha hydroxy 6 beta meXrenone; (g) 9 11 alpha hydroxy testosterone; and (k) progesterone to 11 alpha meXrenone to 11 alpha hydroxy 9 alpha meXrenone; alpha hydroxy progesterone; (1) meXrenone 6,7-bis-lactone (h) 12 beta mexrenone to 11 alpha hydroxy 12 beta to 11 alpha hydroxy meXrenone 6,7-bis-lactone; and (m) meXrenone; (ii) delta 12 meXrenone to 11 alpha hydroxy meXrenone 7.9-bislactone to 11 alpha hydroxy meXrenone delta 12 meXrenone; (ii) testosterone to 11 alpha hydroxy 7.9-bislactone and wherein the heterologous DNA is oper testosterone; (k) progesterone to 11 alpha hydroxy proges ably linked to control Sequences required to express the terone; (1) meXrenone 6,7-bis-lactone to 11 alpha hydroxy encoded enzymes in a recombinant host. Preferably the meXrenone 6,7-bis-lactone; and (m) meXrenone 7.9-bislac heterologous DNA coding Sequences in the expression cas tone to 11 alpha hydroxy mexrenone 7.9-bislactone. More Sette are Selected from the group consisting of the following preferably, the hydroxylation is selected from the group genus and Species: Aspergillus Ochraceus, Aspergillus consisting of: (a) canrenone to 11 alpha hydroxy canrenone; Ochraceus, Aspergillus niger, Aspergillus nidulans, Rhizopus (b) androstenedione to 11 alpha hydroxy androstenedione; Oryzae, Rhizopus Stolonifer, Streptomyces fradiae, Bacillus (c) aldona to 11 alpha hydroxy aldona; and (d) ADD (1,4 megaterium, Pseudomonas cruciviae, Trichothecium androstenedienedione) to 11 alpha hydroxy ADD. Most rO.Seum, Fusarium Oxysporum Rhizopus arrhizus, Absidia preferably the hydroxylation is from canrenone to 11 alpha coerula, Absidia glauca, Actinomucor elegans, Aspergillus hydroxy canrenone. flavipes, Aspergillus fumigatus, Beauveria bassiana, Botry 0024. The invention also provides for an expression OSphaeria Obtusa, Calonectria decora, Chaetomium cassette comprising a promoter operably linked to an iso cochliodes, CorynespOra cassicola, Cunninghamella lated and purified nucleic acid encoding a polypeptide that blakesleeana, Cunninghamella echinulata, Cunninghamella can catalyze the 11 alpha hydroxylation of 3 keto delta 4.5 elegans, Curvularia clavata, Curvularia lunata, Cylindro steroids (3 keto delta 4 steroids); 3 keto delta 4, 5 delta 6,7 carpOn radicicola, EpicOccum humicola, GongrOnella but steroids (3 keto delta 4 delta 6 steroids); 3 keto delta 6, 7 leri, Hypomyces chrySOSpermus, Monosporium Olivaceum, steroids (3 keto delta 6 steroids); or 3 keto delta 1, 2 delta Mortierella isabellina, Mucor mucedo, Mucor griseocyanus, 4, 5 steroids (3 keto delta 1 delta 4 steroids). More prefer Myrothecium verrucaria, Nocardia corallina, Paecilomyces ably, the hydroxylation is Selected from the group consisting carneus, Penicillum patulum, Pithomyces atroolivaceus, of: (a) canrenone to 11 alpha hydroxy canrenone; (b) andros Pithomyces cynodontis, Pycnosporiuin sp., Saccha tenedione to 11 alpha hydroxy androstenedione; (c) aldona ropolyspora erythrae, Sepedonium chrySOSpermum, Stachy to 11 alpha hydroxyaldona; (d) ADD (1,4 androstenediene lidium bicolor, Streptomyces hygroscopicus, Streptomyces dione) to 11 alpha hydroxy ADD; (e) mexrenone to 11 alpha purpuraScens, SyncephalaStrum racemosum, ThamnoStylum hydroxy meXrenone, (f) 6 beta mexrenone to 11 alpha piriforme, Thielavia terricola, and Verticillium theobromae, hydroxy 6 beta meXrenone; (g) 9 alpha meXrenone to 11 Cephalosporium aphidicola, Cochliobolus lunatas, alpha hydroxy 9 alpha meXrenone; (h) 12 beta meXrenone to Tieghemella Orchidis, Tieghemella hyalospora, Monospo 11 alpha hydroxy 12 beta mexrenone; (ii) delta 12 meXrenone rium Olivaceum, Aspergillus ustus, Fusarium graminearum, to 11 alpha hydroxy delta 12 meXrenone; () testosterone to Verticillium glaucum, and Rhizopus nigricans. More prefer 11 alpha hydroxy testosterone; (k) progesterone to 11 alpha ably, the genus and Species are Selected from the group hydroxy progesterone; (1) meXrenone 6,7-bis-lactone to 11 consisting of Aspergillus Ochraceus, Aspergillus Ochraceus, alpha hydroxy meXrenone 6,7-bis-lactone; and (m) Aspergillus niger, Aspergillus nidulans, Rhizopus Oryzae, meXrenone 7.9-bislactone to 11 alpha hydroxy meXrenone Rhizopus Stolonifer, Streptomyces fradiae, Bacillus megate 7.9-bislactone. More preferably, the hydroxylation is rium, Pseudomonas cruciviae, Trichothecium roseum, Selected from the group consisting of: (a) canrenone to 11 Fusarium Oxysporum, Rhizopus arrhizus, and Monosporium alpha hydroxy canrenone; (b) androstenedione to 11 alpha Olivaceum. Most preferably, genus and Species is Aspergillus hydroxy androstenedione; (c) aldona to 11 alpha hydroxy Ochraceus. aldona; and (d) ADD (1,4 androstenedienedione) to 11 alpha 0027 Preferably, the recombinant host cell and progeny hydroxy ADD. Most preferably the hydroxylation is from thereof comprise at least one expression cassette. More canrenone to 11 alpha hydroxy canrenone. preferably, the host is a microorganism. Most preferably, the US 2005/0003473 A1 Jan. 6, 2005 host is a bacterium. The invention also provides for a proceSS 1 delta 4 steroids) wherein the hydroxylation is selected for making one or more enzymes from the metabolic path from the group consisting of: (a) canrenone to 11 alpha way for the transformation of Sitosterol to eplerenone com hydroxy canrenone; (b) androstenedione to 11 alpha prising incubating the recombinant host cell in a nutrient hydroxy androstenedione; (c) aldona to 11 alpha hydroxy medium under conditions where the one or more enzymes aldona; (d) ADD (1,4 androstenedienedione) to 11 alpha encoded by the heterologous DNA are expressed and accu hydroxy ADD; (e) mexrenone to 11 alpha hydroxy mulate. More preferably the proceSS comprises the Steps of meXrenone, (f) 6 beta meXrenone to 11 alpha hydroxy 6 beta (a) incubating the compound to be oxidized in the presence meXrenone; (g) 9 alpha meXrenone to 11 alpha hydroxy 9 alpha meXrenone; (h) 12 beta mexrenone to 11 alpha the recombinant host cells under conditions where the hydroxy 12 beta meXrenone; (ii) delta 12 meXrenone to 11 compound is hydroxylated and the hydroxylated product alpha hydroxy delta 12 meXrenone; () testosterone to 11 accumulates, and (b) recovering the hydroxylated product. alpha hydroxy testosterone; and (k) progesterone to 11 alpha Most preferably, the process comprises the steps of: (a) hydroxy progesterone. Preferably the enzyme does not cata incubating the compound to be oxidized in the presence of lyze the 15 beta hydroxylation of 3 keto delta 4.5 steroids; the enzymes produced under conditions where the com 3 keto delta 4, 5 delta 6, 7 steroids; or 3 keto delta 6, 7 pound is hydroxylated and the hydroxylated product accu Steroids. mulates, and (b) recovering the hydroxylated product. The invention also provides for a host cells harboring an expres 0031. The invention also provides for a purified polypep Sion cassette. More preferably the expression cassette is tide, the amino acid Sequence of which is Selected from the integrated into the chromosome of Said host cell. More group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ preferably, the expression cassette is integrated into an ID NO: 25. expression vector. 0032. The invention provides for a purified immunogenic polypeptide, the amino acid Sequence of which comprises at 0028. The invention also provides for a method of deter mining the Specific activity of a cloned 11 alpha hydroxylase least ten consecutive residues of SEQ ID NO: 2. comprising the steps of: (a) transforming host cells with an 0033. The invention provides for an isolated and purified expression vector comprising a nucleic acid that encodes antibody having a binding Specificity for 11 alpha hydroxy said 11 alpha hydroxylase, (b) expressing said 11 alpha lase having an amino acid Sequence as shown in SEQ ID hydroxylase in Said host cells; (c) preparing Subcellular NO: 2. Preferably the antibody binds to a protein region membrane fractions from said cells, (d) incubating said Selected from the group consisting of (a) the N-terminal Subcellular membrane fractions with a Steroid Substrate, and amino acids 1-10 of SEQ ID NO: 2; (b) the last 10 (e) monitoring conversion of the Steroid Substrate to its 11 C-terminal amino acids of SEQ ID NO: 2; (c) amino acids alpha hydroxy steroid counterpart. Preferably, the further SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and (e) comprises transforming host cells with an expression vector amino acids SEQ ID NO: 25. Preferably the antibody is nucleic acid that encodes an oxidoreductase, and expressing purified on a peptide column, wherein Said peptide is Said oxidoreductase in Said host cells. More preferably, the Selected from the group consisting of: (a) the N-terminal oxidoreductase is human or Aspergillus Ochraceus. Most amino acids 1-10 of SEQ ID NO: 2; (b) the last 10 preferably the oxidoreductase is human oxidoreductase. C-terminal amino acids of SEQ ID NO: 2; (c) amino acids Most preferably the oxidoreductase is Aspergillus Ochraceus SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and (e) oxidoreductase. amino acids SEO ID NO: 25. 0029. The invention also provides for a protein having 0034. The invention also provides for a purified polypep SEO ID NO: 2 and variants thereof that are at least 95% tide, the amino acid Sequence of which is Selected from the identical to SEQ ID NO: 2 and catalyze the 11 alpha group consisting of SEQ ID NO: 26. hydroxylation of 3 keto delta 4, 5 steroids; 3 keto delta 4, 5 delta 6, 7 steroids; 3 keto delta 6, 7 steroids; or 3 keto delta 0035. The invention also provides for a purified immu 1, 2 delta 4, 5 Steroids, wherein Said hydroxylation is nogenic polypeptide, the amino acid Sequence of which Selected from the group consisting of: (a) canrenone to 11 comprises at least ten consecutive residues of SEQ ID NO: alpha hydroxy canrenone; (b) androstenedione to 11 alpha 6. hydroxy androstenedione; (c) aldona to 11 alpha hydroxy 0036) The invention also provides for an isolated and aldona; (d) ADD (1,4 androstenedienedione) to 11 alpha purified antibody having a binding Specificity for 11 alpha hydroxy ADD; (e) mexrenone to 11 alpha hydroxy hydroxylase having an amino acid Sequence as shown in meXrenone, (f) 6 beta meXrenone to 11 alpha hydroxy 6 beta SEQ ID NO: 6. Preferably the antibody binds to a protein meXrenone; (g) 9 alpha meXrenone to 11 alpha hydroxy 9 region selected from the group consisting of (a) the N-ter alpha meXrenone; (h) 12 beta mexrenone to 11 alpha minal amino acids 1-10 of SEQ ID NO: 6; (b) the last 10 hydroxy 12 beta meXrenone; (ii) delta 12 meXrenone to 11 C-terminal amino acids of SEQ ID NO: 6; and (c) amino alpha hydroxy delta 12 meXrenone; () testosterone to 11 acids SEQ ID NO: 26. More preferably, the antibody is alpha hydroxy testosterone; and (k) progesterone to 11 alpha purified on a peptide column, wherein Said peptide is hydroxy progesterone. Preferably the enzyme does not cata Selected from the group consisting of: (a) the N-terminal lyze the 15 beta hydroxylation of 3 keto delta 4.5 steroids; amino acids 1-10 of SEQ ID NO: 6; (b) the last 10 3 keto delta 4, 5 delta 6, 7 steroids; or 3 keto delta 6, 7 C-terminal amino acids of SEQ ID NO: 6; and (c) amino Steroids. acids SEO ID NO: 26. 0030 The invention provides an isolated and purified 0037. The invention also provides for a composition nucleic acid encoding an enzyme that can catalyze the 11 comprising an antibody described above in an effective alpha hydroxylation of 3 keto delta 4.5 steroids (3 keto delta carrier, vehicle, or auxiliary agent. It also provides for a 4 steroids); 3 keto delta 4, 5 delta 6, 7 steroids (3 keto delta composition comprising Such an antibody and a Solution. 4 delta 6 steroids); 3 keto delta 6, 7 steroids (3 keto delta 6 The antibody may be a polyclonal antibody. The antibody steroids); or 3 keto delta 1, 2 delta 4, 5 steroids (3 keto delta may also be a monoclonal antibody. The antibody may be US 2005/0003473 A1 Jan. 6, 2005 conjugated to an immunoaffinity matrix. The invention also 0046) The invention also provides for a DNA construct provides for a method of using an immunoaffinity matrix to which alters the expression of a 11 alpha hydroxylase gene purify a polypeptide from a biological fluid or cell lysate. not normally expressed in a cell when said DNA construct Preferably the immunoaffinity matrix is SEPHAROSE 4B. is inserted into chromosomal DNA of the cell, said DNA More preferably the method of using an immunoaffinity construct comprising: (a) a targeting Sequence; (b) a regu matrix to purify a polypeptide from a biological fluid or cell latory Sequence; and (c) the structural gene for a steroid lysate uses SEPHAROSE 4B as an immunoaffinity matrix. oxidoreductase. The invention also provides for a host cell More preferably, the method of using an immunoaffinity harboring this DNA construct. matrix to purify a polypeptide from a biological fluid or cell 0047 The invention also provides for use of a host cell lysate uses SEPHAROSE 4B as an immunoaffinity matrix. harboring a cloned 11 alpha hydroxylase for the manufacture 0.038. The invention also provides for a method of using of a medicament for therapeutic application to treat heart a peptide column to purify an antibody, wherein Said peptide disease, inflammation, arthritis, or cancer. is selected from the group consisting of: (a) the N-terminal 0048. The invention also provides for a composition amino acids 1-10 of SEQ ID NO: 2; (b) the last 10 comprising from about 0.5-to about 500 g/L molasses, C-terminal amino acids of SEQ ID NO: 2; (c) amino acids 0.5-50 g/L cornsteep liquid, 0.5-50 g/L KHPO, 2.5-250 SEQ ID NO: 23; (d) amino acids SEQ ID NO: 24; and (e) g/L NaCl, 2.5-250 g/L glucose, and 0.04-4 g/L progesterone, amino acids SEO ID NO: 25. pH 3.5-7. Preferably, this composition is comprised of from about 10-250 g/L molasses, 1-25 g/L cornsteep liquid, 1-25 0.039 The invention also provides for a method of using g/L KHPO, 5-125 g/L NaCl, 5-125 g/L glucose, and 0.08-2 a peptide column to purify an antibody, wherein Said peptide g/L progesterone, pH 4.5-6.5. More preferably, the compo is selected from the group consisting of: (a) the N-terminal sition is comprised of from about 25-100 g/L molasses, amino acids 1-10 of SEQ ID NO: 6; (b) the last 10 2.5-10 g/L cornsteep liquid, 2.5-10 g/L KHPO, 12.5-50 C-terminal amino acids of SEQ ID NO: 6; and (c) amino g/L NaCl, 12.5-50 g/L glucose, and 0.2-0.8 g/L progester acids SEO ID NO: 26. one, pH 5.5-6.0. Most preferably the composition comprises 0040. The invention also provides for a method of detect about 50 g/L molasses, 5 g/L cornsteep liquid, 5 g/L ing a first polypeptide in a biological fluid, wherein Said first KHPO, 25 g/L NaCl, 25 g/L glucose, 20 g/L agar, and 0.4 polypeptide is Selected from the group consisting of 11 alpha g/L progesterone, pH 5.8. hydroxylase and oxidoreductase, comprising the following 0049. The invention also provides for a semisolid formu steps: (a) contacting said fluid with a second polypeptide, lation of any of the compositions described above, further having a binding Specificity for Said first polypeptide, and comprising from about 4-100 g/L agar. Preferably the agar (b) assaying the presence of Said Second polypeptide to is at a concentration of from about 10-40 g/L agar. More determine the level of said first polypeptide. Preferably, the preferably, the agar is about 20 g/L agar. Second polypeptide is an antibody. More preferably, the 0050. The invention also provides for the use of any of Second polypeptide is radiolabeled. the compositions describe above to produce Spores from the 0041. The invention also provides for a process for microorganism Selected from the group consisting of producing an isolated nucleic acid comprising hybridizing Aspergillus Ochraceus, Aspergillus niger, Aspergillus nidu lans, Rhizopus Oryzae, Rhizopus Stolonifer, and Trichoth SEQ ID NO: 1 to genomic DNA in 6xSSC and 65° C. and ecium roseum, Fusarium Oxysporum Rhizopus arrhizus, isolating the nucleic acid detected with SEQ ID NO: 1. The Monosporium Olivaceum. Penicillum chrysogenium, and invention also provides for an isolated DNA nucleic acid Absidia coerula. Preferably, the composition is used to prepared according to this process. produce Spores from Aspergillus Ochraceus. 0042. The invention also provides for an isolated nucleic acid that Specifically hybridizes under highly Stringent con Definitions ditions to the complement of the sequence set forth in SEQ ID NO: 1. 0051. The following is a list of abbreviations and the corresponding meanings as used interchangeably herein: 0043. The invention also provides for a process for producing an isolated nucleic comprising hybridizing SEQ 0052 11 alpha hydroxycanrenone=11 alpha ID NO: 5 to genomic DNA in 6xSSC and 65° C. and hydroxy-4-androstene-3,17-dione (C.H.O., MW isolating the nucleic acid detected with SEQ ID NO: 5. The 356.46) invention also provides for an isolated DNA nucleic acid prepared according to this process. 0053 AcNPV=Autographa californica nuclear polyhedrosis virus, a member of the Baculoviridae 0044) The invention also provides for an isolated nucleic family of insect viruses acid that Specifically hybridizes under highly Stringent con ditions to the complement of the sequence set forth in SEQ 0054 AD=androstenedione or 4-androstene-3,17 ID NO: 5. dione (CHO, MW 340.46) 004.5 The invention also provides for a DNA construct 0055 aldadiene=canrenone which alters the expression of a 11 alpha hydroxylase gene not normally expressed in a cell when said DNA construct 0056 Amp=ampicillin is inserted into chromosomal DNA of the cell, said DNA construct comprising: (a) a targeting Sequence; (b) a regu 0057 attTn7=attachment site for TnT (a preferential latory Sequence; and (c) the structural gene for a steroid 11 site for TnT insertion into bacterial chromosomes) alpha hydroxylase. The invention also provides for a host 0058 bacmid=recombinant baculovirus shuttle vec cell harboring this DNA construct. tor isolated from E. coli US 2005/0003473 A1 Jan. 6, 2005
0059 Bluo-gal=halogenated indolyl-B-D-galacto 0096 X-gal=5-bromo-3-chloro-indolyl-f-D-galac Side topyranoside 0060 bp=base pair(s) 0097. X-gluc=5-bromo-3-chloro-indolyl-B-D-glu copyranoside 0061 Cam=chloramphenicol 0098. The following is a list definitions of various terms 0062) cDNA=complementary DNA used herein: 0063) DMF=N,N-dimethylformamide 0099] The species cAspergillus ochraceus NRRL 405” means the filamentous fungus Aspergillus Ochraceus NRRL 0064 ds=double-stranded 405, accession number 18500, obtained from the American 0065 eplerenone or epoxymexrenone=methyl Type Culture Collection (ATCC). A. ochraceus NRRL 405 hydrogen 9,11C.-epoxy-17C-hydroxy-3-oxopregn-4- and A. OchraceuS ATCC 18500 are the same strain, cata ene-7C,21-dicarboxylate, Y-lactone (MW 414.5) logued differently. 0100 The term “amino acid(s)" means all naturally 0066 g=gram(s) occurring L-amino acids, including norleucine, norvaline, 0067 Gen=gentamicin homocysteine, and ornithine. 0068 hoXr=human oxidoreductase 0101 The term “degenerate” means that two nucleic acid molecules encode for the same amino acid Sequences but 0069. HPLC=high performance liquid chromatogra comprise different nucleotide Sequences. phy 0102) The term “fragment” means a nucleic acid mol 0070 hydroxycanrenone=11 alpha- or 11 beta-hy ecule whose Sequence is shorter than the target or identified droxycanrenone nucleic acid molecule and having the identical, the Substan tial complement, or the Substantial homologue of at least 10 0071 IPTG=isopropyl-f-D-thiogalactopyranoside contiguous nucleotides of the target or identified nucleic 0072 Kan=kanamycin acid molecule. 0103) The term “fusion protein' means a protein or 0.073 kb=kilobase(s), 1000 bp(s) fragment thereof that comprises one or more additional 0074 mb=megabase(s) peptide regions not derived from that protein. 0075 Me=methyl 0104. The term “probe” means an agent that is utilized to determine an attribute or feature (e.g. presence or absence, 0076 mg=milligram(s) location, correlation, etc.) of a molecule, cell, tissue, or 0.077 ml or mL=milliliter(s) organism. 0105 The term “promoter” is used in an expansive sense 0078 mm=millimeter to refer to the regulatory Sequence(s) that control mRNA 0079 mM=millimolar production. Such Sequences include RNA polymerase bind ing sites, enhancers, etc. 0080) NMR=nuclear magnetic resonance 0106 The term “protein fragment” means a peptide or 0081 oxr=oxidoreductase polypeptide molecule whose amino acid Sequence com 0082 PCR=polymerase chain reaction prises a Subset of the amino acid Sequence of that protein. 0107 The term “recombinant” means any agent (e.g., 0083 r=resistant or resistance DNA, peptide, etc.), that is, or results from, however indi 0084) RP-HPLC=reverse phase high performance rectly, human manipulation of a nucleic acid molecule. liquid chromatography 0108. The term “selectable or screenable marker genes' 0085 RT=room temperature means genes whose expression can be detected by a probe 0.086 RT-PCR=reverse transcriptase polymerase as a means of identifying or Selecting for transformed cells. chain reaction 0109 The term “specifically bind” means that the binding of an antibody or peptide is not competitively inhibited by 0087 S=Sensitive the presence of non-related molecules. 0088 SDS-PAGE=sodium dodecyl sulfate poly 0110. The term “specifically hybridizing” means that two acrylamide gel electrophoresis nucleic acid molecules are capable of forming an anti 0089 Spc/Str=spectinomycin/streptomycin parallel, double-Stranded nucleic acid structure. 0090 Tet=tetracycline 0111. The term “substantial complement” means that a nucleic acid Sequence shares at least 80% sequence identity 0091 Tn=transposon with the complement. 0092 ts=temperature-sensitive 0112 The term “substantial fragment” means a nucleic 0.093 U=units acid fragment which comprises at least 100 nucleotides. 0113. The term “substantial homologue” means that a 0094) ug or lug=microgram(s) nucleic acid molecule shares at least 80% sequence identity 0.095 ul or ul-microliter(s) with another. US 2005/0003473 A1 Jan. 6, 2005
0114. The term “substantially hybridizing” means that 0115 The term “substantially-purified” means that one or two nucleic acid molecules can form an anti-parallel, more molecules that are or may be present in a naturally double-Stranded nucleic acid Structure under conditions occurring preparation containing the target molecule will (e.g., Salt and temperature) that permit hybridization of have been removed or reduced in concentration. Sequences that exhibit 90% sequence identity or greater with each other and exhibit this identity for at least about a 0116. The following is a list of steroids, corresponding contiguous 50 nucleotides of the nucleic acid molecules. terms, and their Structures, as used interchangeably herein:
# Name CA Index Name: Other Names Formula Structure
1. Eplerenone Pregn-4-ene-7,21- Spiro 9,11-epoxy-9H- C24H3OO6 dicarboxylic acid, cyclopentalaphenanthrene 9,11-epoxy-17-hydroxy- 17(2H).2'(3'H)-furan, 3-oxo-y-lactone.methyl pregn-4-ene-7,21 ester,(7c,11C,17Cl)-(9CI) dicarboxylic acid deriv.; CGP30083: Eplerenone; SC66110
2 Aldadiene; Pregna-4,6-diene-21- 17C-Pregna-4,6-diene-21- C22H28O3 Canrenone carboxylic acid, carboxylic acid, 17 17-hydroxy-3-oxo-, hydroxy-3-oxo-, y-lactone y-lactone, (17Cl)-(9CI) (6CI, 7GI, 8CI):Spiro17H cyclopentalaphenanthrene 17.2"(5"H)-furan, pregna-4, 6-diene-21-carboxylic acid deriv.: 11614.R.P.; 17B Hydroxy-3-oxopregna-4,6- diene-21-carboxylic acid; 20-Spiroxa-4,6-diene-3, 21-dione; Aldadiene; Canremone: Phanurane: SC9376; Spirolactone SC14266
3 1160 - Pregna-4,6-diene-21- 11C-Hydroxycan renone C22H28O4 Hydroxycan renone carboxylic acid, 11,17 dihydroxy-3-oxo-, Y lactone,(11C,17Cl)-(9CI)
5 Aldona ethyl Pregna-4,6-diene-21- Spiro17H- C24H34O3 enol ether carboxylic acid, 3-ethoxy- cyclopentalaphenanthrene 17-hydroxy-, 17,2'5"H)-furan, pregna y-lactone (9CI) 4,6-diene-21-carboxylic acid deriv.; Aldona ethyl enol ether
EtO
US 2005/0003473 A1 Jan. 6, 2005
-continued
# Name CA Index Name: Other Names Formula Structure
16 11O- Androsta-1,4-diene-3, Androsta-1,4-diene-3, C19H24O3 Hydroxyandrosta- 17-dione, 11-hydroxy-, 17-dione, 11c-hydroxy 1,4-diene-3,17 (6CI, 7GI, 8CI); 11c dione (11 alpha Hydroxyandrosta-1,4-diene hydroxy ADD) 3,17-dione: Kurchinin
17 aldona Compound 5 (aldonaethyl enol ether) with O = in place of EtO- at position 3 18 eXCOe Compound 12 with cyclic bis-lactone 6,7-bislactone ring (-O-C=O-) formed between carbons at positions 6 and 7 (See US 5,981,744 for discussion of similar lactone rings) 19 11 alpha 11 alpha hydroxy version of hydroxy Compound 18 eXCOe 6,7-bislactone 2O eXCOe Compound 11 with cyclic bis-lactone 7.9-bislactone ring (-O-C=O-) formed between carbons at positions 7 and 9 (See US 5,981,744 for discussion of similar lactone rings) 21 11 alpha 11 alpha hydroxy version of hydroxy Compound 20 eXCOe 7.9-bislactone
0117 FIG. 1-Nucleotide and protein sequence of 0122) References for these loci are as follows: A60557 Aspergillus OchraceuS 11 alpha hydroxylase Yamano, S., Aoyama, T., McBride, O.W., Hardwick, J. P., Gelboin, H. V. and Gonzalez, F. J. Human NADPH-P450 0118. The nucleotide and protein sequences of Aspergil oxidoreductase: complementary DNA cloning, Sequence lus ochraceus 11 alpha hydroxylase (SEQ ID NO: 1, SEQID and vaccinia virus-mediated expression and localization of NO: 2, respectively) are displayed. the CYPOR gene to chromosome 7 Mol. Pharmacol. 36 (1), 83-88 (1989); AAG09798 Czerwinski, M., Sahni, M., 0119 FIG. 2-Nucleotide and protein sequence of Madan, A. and Parkinson, A. Polymorphism of human human oxidoreductase CYPOR: Expression of new allele. Unpublished, Direct Submission, and P16435 Haniu, M., McManus, M. E., 0120) The nucleotide and protein sequences of human Birkett, D. J., Lee, T. D. and Shively, J. E. Structural and oxidoreductase (SEQ ID NO:3, SEQ ID NO: 4, respec functional analysis of NADPH-cytochrome P-450 reductase tively) are displayed. The predicted amino acid sequence of from human liver: complete Sequence of human enzyme and human oxidoreductase independently cloned from a cDNA NADPH-binding sites. Biochemistry 28 (21), 8639-8645 library prepared by RT-PCR using the RNA from a human (1989); AAB21814Shephard, E. A., Palmer, C. N., Segall, HepG2 cells as a template, as disclosed in this specification, H. J. and Phillips, I. R. Quantification of cytochrome P450 matches that previous reported by three different laborato reductase gene expression in human tissues. Arch. Biochem. ries. The GenBank accession numbers for these loci include Biophys. 294 (1), 168-172 (1992); S90469 Shephard, E. A60557 (NADPH-ferrihemoprotein reductase (EC A., Palmer, C. N., Segall, H. J. and Phillips, I. R. Quanti 1.6.2.4)-human); AAG09798 (NADPH-cytochrome P450 fication of cytochrome P450 reductase gene expression in reductase Homo sapiens), and P16435 (NADPH-CYTO human tissues. Arch. Biochem. Biophys. 294 (1), 168-172 CHROME P450 REDUCTASE (CPR) (P450R)). (1992)). 0123 FIG. 3-Nucleotide and protein sequence of 0121 The amino acid sequence of AAB21814 (cyto Aspergillus Ochraceus oxidoreductase chrome P450 reductase EC 1.6.2.41 human, placenta, Peptide Partial, 676 aa), differs from human oxidoreductase 0.124. The nucleotide and protein sequences of Aspergil A60557 and P16435 at 4 residues: A->V at 500, F->L at lus ochraceus 11 oxidoreductase (SEQ ID NO: 5, SEQ ID 518, V->W at 537, and A->H at 538. The initial methionine NO: 6, respectively) are displayed. is also missing from AAB21814. The cognate nucleic acid 0.125 FIG. 4-Amino acid homology alignment of A. for AA21814 (S90469 cytochrome P450 reductase human, Ochraceus 11 alpha hydroxylase with the top 10 BLAST hits placenta, mRNA Partial, 2403 nt) lacks the ATG codon for from GenBank the initial methionine and includes a C->T change at 1496, 0.126 Aspergillus Ochraceus steroid 11 alpha hydroxy a C->A, change at 1551, and a frameshift due to a missing lase (SEQ ID NO: 02), cloned into plasmid pMON45624 G at 1605 which is resolved by the addition of a T at 1616. (SEQ ID NO: 01), was aligned with related enzymes found US 2005/0003473 A1 Jan. 6, 2005 in GenBank using the BLASTP program that implements a 0.130 FIG. 6-Percent homology between Aspergillus heuristic matching algorithm (Altschul et al., J Mol Biol Ochraceus 11 alpha hydroxylase and the top 10 BLAST hits October 5:215(3):403-10, 1990). The GenBank accession from GenBank numbers (its probable function, genus and species) for the top 10 matches are as follows: CAA75565 (cytochrome 0131 The percent homology between Aspergillus Ochra P450 monooxygenase Gibberella fujikuroil; CAB91316 ceuS Steroid 11. alpha hydroxylase and the top 10 enzymes (probable cytochrome P450 monooxygenase (lovA) Neu found in GenBank using BLAST was calculated using rospora crassa); CAB56503 (cytochrome P450 Catharan CLUSTAL (Thompson et al., Comput. Appl. BioSci. 10:19 thus roseus); AAB94588 (CYP71D10p Glycine max); 29, 1994). CAA75566 (cytochrome P450 monooxygenase Gibberella fujikuroi); AAD34552 (cytochrome P450 monooxygenase 0132 FIG. 7-Amino acid homology alignment of Aspergillus terreus); CAA75567 (cytochrome P450 Aspergillus OchraceuS and human oxidoreductase to monooxygenase Gibberella fujikuroi); CAA76703 (cyto NADPH cytochrome P450 reductases from A. niger, mouse, chrome P450 Gibberella fujikuroil); CAA57874 (unnamed and S. cerevisiae protein product Fusarium oxysporum); CAA91268 (simi 0133. The amino acid sequences of Aspergillus Ochra lar to cytochrome P450-cDNAESTyk423b11.3 comes from ceus steroid oxidoreductase (SEQ ID NO: 06) cloned into this gene Caenorhabditis elegans). plasmid pMON45632 (SEQ ID NO: 05), and human oxi 0127. References for these loci are as follows: doreductase (SEQ ID NO: 03), cloned into plasmid CAA75565 Tudzynski, B. and Holter, K., Gibberellin bio pMON45605 (SEQ ID NO: 04) were aligned with related synthetic pathway in Gibberella fujikuroi evidence for a enzymes from A. niger, mouse, and S. cervisiase, as gene cluster. Fungal Genet. Biol. 25 (3), 157-170 (1998); described above. The GenBank accession numbers (prob CAB91316 Schulte, U., Aign, V., Hoheisel, J., Brandt, P., able function, genus and species) are as follows: Fartmann, B., Holland, R., Nyakatura, G., Mewes, H.W. and BAAO2936 (NADPH-cytochrome P450 reductase precursor Mannhaupt, G., Unpublished; CAB56503 Schroeder, G., Saccharomyces cerevisiae); CAA81550 NADPH cyto Unterbusch, E., Kaltenbach, M., Schmidt, J., Strack, D. and chrome P450 oxidoreductase Aspergillus niger; P16435 Schroeder, J. Light-induced cytochrome P450-dependent (NADPH-CYTOCHROME P450 REDUCTASE (CPR) enzyme in indole alkaloid biosynthesis: taberSonine 16-hy (P450R) human); BAAO4496 (NADPH-cytochrome P450 droxylase FEBS Lett. 458, 97-102 (1999)); AAB94588 oxidoreductase Mus musculus). Siminszky, B., Corbin, F.T., Ward, E. R., Fleischmann, T. J. and Dewey, R. E. Expression of a Soybean cytochrome 0134) References for these loci are as follows: P450 monooxygenase cDNA in yeast and tobacco enhances BAAO2936 Yabusaki, Y., Murakami, H. and Ohkawa, H. the metabolism of phenylurea herbicides. Proc. Natl. Acad. Primary structure of Saccharomyces cerevisiae NADPH Sci. U.S.A. 96 (4), 1750-1755 (1999)); CAA75566 Tudzyn cytochrome P450 reductase deduced from nucleotide ski, B. and Holter, K. Gibberellin biosynthetic pathway in sequence of its cloned gene. J. Biochem. 103 (6), 1004-1010 Gibberella fujikuroi: evidence for a gene cluster. Fungal (1988); CAA81550 van den Brink, J., van Zeijl, C., van Genet. Biol. 25 (3), 157-170 (1998); AAD34552 Kennedy, den Hondel, C. and Van Gorcom, R. Cloning and charac J., Auclair, K., Kendrew, S. G., Park, C., Vederas, J. C. and terization of the NADPH cytochrome P450 oxidoreductase Hutchinson, C. R. Accessory Proteins Modulate Polyketide (cprA) gene of Aspergillus niger. Unpublished); P16435 Synthase Activity During Lovastatin Biosynthesis. Science (1999) In press); CAA75567 Tudzynski, B. and Holter, K. Haniu, M., McManus, M. E., Birkett, D. J., Lee, T. D. and Gibberellin biosynthetic pathway in Gibberella fujikuroi: Shively, J. E. Structural and functional analysis of NADPH evidence for a gene cluster. Fungal Genet. Biol. 25 (3), cytochrome P-450 reductase from human liver: complete 157-170 (1998); CAA76703 Tudzynski, B. and Hoelter, K. sequence of human enzyme and NADPH-binding sites Bio Characterization of P450 monooxygenase genes from Gib chemistry 28 (21), 8639-8645 (1989); BAA04496 Ohgiya, berella fujikuroi. Unpublished; CAA57874 Mouyna, I. and S., Shinriki, N., Kamataki, T. and Ishizaki, K. Mouse Brygoo, Y. Disruption of a Fusarium Oxysporum f.sp. elaei NADPH-cytochrome P-450 oxidoreductase: molecular dis cytochrome P450 gene by a repetitive Sequence. Unpub cloning and functional expression in yeast. Biochim. Bio lished; and CAA91268 No Authors. Genome sequence of phys. Acta 1186 (1-2), 137-141 (1994). the nematode C. elegan.S. a platform for investigating biol ogy. The C. elegans Sequencing Consortium. Science 282 0.135 FIG. 8-Amino acid homology alignment of A. (5396), 2012-2018 (1998) Published errata appear in Sci Ochraceus oxidoreductase to NADPH cytochrome P450 ence Jan. 1, 1999;283(5398):35 and Mar. 26, reductases from A. niger, mouse, and S. cerevisiae 1999;283(5410):2103 and Sep. 3, 1999:285(5433): 1493). 0.136 The amino acid sequence of Aspergillus ochraceus steroid oxidoreductase (SEQ ID NO: 06) cloned into plas 0128 FIG. 5-Phylogenetic tree showing the relatedness mid pMON45632 (SEQ ID NO: 05), was aligned with of Aspergillus Ochraceus 11 alpha hydroxylase to the top 10 related fungal enzymes from A. niger and S. cervisiase, as BLAST hits from GenBank described above. Descriptions of the GenBank accession 0129. A phylogenetic tree displaying the genetic related numbers used as labels in the figure are the same as that neSS of Aspergillus Ochraceus Steroid 11 alpha hydroxylase, described above for the legend to FIG. 7, above. cloned into plasmid pMON45624, was aligned with related 0.137 FIG. 9-Phylogenetic tree showing the relatedness enzymes found in GenBank. BLAST was used to find the related enzymes within GenBank, and ClustalW was used of Aspergillus OchraceuS and human oxidoreductase to generate the multiple Sequence alignment and phylogenetic reductases from A. niger, yeast, and mouse. tree depicted in this figure. Descriptions of the GenBank 0.138 A phylogenetic tree displaying the genetic related accession numbers used as labels in the figure are the same ness of Aspergillus Ochraceus oxidoreductase (SEQ ID NO: as that described above for the legend to FIG. 4. 06), cloned into plasmid pMON45632 (SEQ ID NO: 05), US 2005/0003473 A1 Jan. 6, 2005
was aligned with related enzymes. BLAST was used to find was used generate the multiple Sequence alignment and the related enzymes within GenBank, and ClustalW was phylogenetic tree depicted in this figure. Descriptions of the used generate the multiple Sequence alignment and phylo SWISPROT accession numbers used as labels in the figure genetic tree depicted in this figure. Descriptions of the are the same as that described above for the legend to FIG. GenBank accession numbers used as labels in the figure are 11, above. the same as that described above for the legend to FIG. 7, above. 0146 FIG. 13-Percent identity between human oxi doreductase and top 4 hits from SwissProt 0139 FIG. 10-Percent identity between Aspergillus Ochraceus oxidoreductase and reductases from A. niger, 0147 The percent identity between human oxidoreduc yeast, and mouse. tase and the top 4 hits found in SWISSPROT was calculated using Clustal W and Boxshade. 0140. The percent identity between Aspergillus Ochra ceus oxidoreductase and the oxidoreductases from A. niger, 0.148 FIG. 14: Expression of Aspergillus ochraceus 11 yeast, and mouse was calculated using Clustal W and alpha hydroxylase in transfected Sf9 insect cells Boxshade. 0149 Baculovirus-infected insect cells expressing 0141 FIG. 11-Alignment of human oxidoreductase Aspergillus OchraceuS 11 alpha hydroxylase were harvested with top 4 hits from SwissProt at 25 and 48 hours post infection and microSomal membrane 0142. The amino acid sequences of human steroid oxi fractions were prepared and Separated by SDS-polyacryla doreductase (SEQ ID NO: 04), cloned into plasmid mide gel electrophoresis. The proteins in the gel were pMON45605 (SEQ ID NO: 03), which corresponds to the electrophoretically transferred to 0.2 um nitrocellulose amino acid Sequence of the corrected Sequence reported for membrane (Schleicher & Schuell Grimsehlstrasse 2337574 P16435 below, was aligned with the top 4 hits from the Einbeck Germany) and probed with antibodies GN-1187 SWISSPROT protein sequence database, as described and GN-1188 prepared from peptide 11aOH peptide 2 above. The SWISSPROT accession numbers locus.com CRQILTPYIHKRKSLKGTTD (SEQ ID NO. 24). mon name and species) probable function) are as follows: P16435 (NCPR HUMAN human NADPH-CYTO 0150 FIG. 15: Expression of Aspergillus ochraceus CHROME P450 REDUCTASE; P00389 (NCPR RABIT} P450 oxidoreductase in transfected Sf9 insect cells rabbit) NADPH-CYTOCHROME P450 REDUCTASE; 0151. Baculovirus-infected insect cells expressing P00388 (NCPR RAT}rat NADPH-CYTOCHROME Aspergillus OchraceuS 11 oxidoreductase were harvested at P450 REDUCTASE; P37040 (NCPR MOUSE-mouse) 25 and 48 hours post infection and microSomal membrane NADPH-CYTOCHROME P450 REDUCTASE; P04175 fractions were prepared and Separated by SDS-polyacryla {NCPR PIG}(pig) (NADPH-CYTOCHROME P450 mide gel electrophoresis. The proteins in the gel were REDUCTASE electrophoretically transferred to 0.2 um nitrocellulose 0143 References for these loci are as follows: P16435 membrane (Schleicher & Schuell Grimsehlstrasse 2337574 Haniu, M., McManus, M. E., Birkett, D. J., Lee, T. D. and Einbeck Germany) and probed with antibodies GN-2023 Shively, J. E. Structural and functional analysis of NADPH and GN-12024 prepared from oxr peptide 1 CTYWAVAKD cytochrome P-450 reductase from human liver: complete PYASAGPAMNG (SEQ ID NO: 26). sequence of human enzyme and NADPH-binding sites. Biochemistry 28 (21), 8639-8645 (1989); P00389 Katagiri, 0152 FIG. 16-Conversion of androstenedione to 11 M., Murakami, H., Yabusaki, Y., Sugiyama, T., Okamoto, alpha hydroxy androstenedione monitored by HPLC M., Yamano, T. and Ohkawa, H. Molecular cloning and sequence analysis of full-length cDNA for rabbit liver 0153 Microsomal and mitochondrial subcellular frac NADPH-cytochrome P-450 reductase mRNA. J. Biochem. tions were prepared from insect cells co-infected with 100 (4), 945-954 (1986); P00388 Porter, T. D. and Kasper, recombinant baculoviruses expressing recombinant C. B. Coding nucleotide sequence of rat NADPH-cyto Aspergillus OchraceuS 11 alpha hydroxylase and human chrome P-450 oxidoreductase cDNA and identification of oxidoreductase cloned from HepG2 cell RNA. The subcel flavin-binding domains. Proc. Natl. Acad. Sci. U.S.A. 82 (4), lular fractions were incubated with 250 uMandrostenedione 973-977 (1985); P37040 Ohgiya, S., Shinriki, N., (AD) in the presence of an NADPH-generating system for Kamataki, T. and Ishizaki, K. Mouse NADPH-cytochrome 120 minutes, and the resulting products were separated by P-450 oxidoreductase: molecular cloning and functional HPLC and monitored by ultraviolet detection at 247 nm. expression in yeast. Biochim. Biophys. Acta 1186 (1-2), Hydroxlase activity was found in the microSomal fraction, 137-141 (1994); P04175 Haniu, M., Iyanagi, T., Miller, P., as expected, but also appeared in the mitochondrial fraction. Lee, T. D. and Shively, J. E. Complete amino acid Sequence These results Suggest that the 11 alpha hydroxylase may of NADPH-cytochrome P-450 reductase from porcine have a tendency to Stick to membranes in disrupted cells, or hepatic microsomes. Biochemistry 25 (24), 7906-7911 that the Separation of the Subcellular fractions in this experi (1986)). ment was insufficient. Panel A illustrates a reaction carried 014.4 FIG. 12-Phylogenetic tree showing the related out using enzyme prepared from a mitochondrial fraction. ness of human oxidoreductases with top 4 hits from Swis The peak in panel A that elutes after AD appears to be S.Prot testosterone. When a microSomal fraction was used, almost as much AD was converted to 11 alpha hydroxy AD, but 0.145) A phylogenetic tree displaying the genetic related relatively more testosterone was also produced. Panel B ness of human oxidoreductase (SEQID NO: 04), cloned into illustrates the same reaction carried out for 120 minutes plasmid pMON45604 (SEQ ID NO: 03), was aligned with without a source of enzyme. Panel C illustrates an HPLC related enzymes found in SWISSPROT. BLAST was used to tracing with 11O-hydroxyandrostenedione Standard added to find the related enzymes within SWISSPROT, and ClustalW incubation buffer. US 2005/0003473 A1 Jan. 6, 2005
DETAILED DESCRIPTION OF THE 13144, Botryosphaeria obtusa IMI 038560, Calonectria INVENTION decora ATCC 14767, Chaetomium cochliodes ATCC 10195, Corynespora cassicola ATCC 16718, Cunninghamella 0154) The present invention encompasses enzymes that blakesleeana ATCC 8688a, Cunninghamella echinulata facilitate the biosynthesis of Steroid molecules, particularly ATCC 3655, Cunninghamella elegans ATCC 9245, Curvu enzymes possessing cytochrome P450 or oxidoreductase laria clavata ATCC 22921, Curvularia lunata ACTT 12071, activities. The present invention is directed, in part, to the Cylindrocarpon radicicola ATCC 1011, Epicoccum humi isolation of a nucleic acid encoding Aspergillus OchraceuS cola ATCC 12722, Gongronella butleri ATCC 22822, Hypo 11 alpha hydroxylase, which exhibits Sequence homology to myces chrysospermus, Mortierella isabellina ATCC 42613, the highly conserved residues that correspond to cytochrome Mucor mucedo ATCC 4605, Mucor griseocyanus ATCC P450 enzymes. It also directed to the isolation of nucleic 1207A, Myrothecium verrucaria ATCC 9095, Nocardia acids encoding human and Aspergillus Ochraceus oxi corallina, Paecilomyces carneus ATCC 46579, Penicillum doreductase. Biological activities of the cloned hydroxylases patulum ATCC 24550, Pithomyces atroolivaceus IFO 6651, and oxidoreductases of the present invention can be deter Pithomyces cynodontis ATCC 26150, Pycnosporium sp. mined by a variety of assays, including incubation of Steroid ATCC 12231, Saccharopolyspora erythrae ATCC 11635, Substrates in the presence of microSomes prepared from Sepedonium chrysospermum ATCC 13378, Stachylidium recombinant baculovirus-infected insect cells and monitor ing the conversion to their 11 alpha hydroxy-counterparts by bicolor ATCC 12672, Streptomyces hygroscopicus ATCC high pressure liquid chromatography (HPLC). The present 27438, Streptomyces purpurascens ATCC 25489, Synceph invention, comprising novel 11 alpha hydroxylase and oxi alaStrum racemosum ATCC 18192, Thamnostylum piri doreductase nucleic acids, proteins, peptides, homologues, forme ATCC 8992, Thielavia terricola ATCC 13807, and and fragments of either, provides new and advantageous Verticillium theobromae ATCC 12474. methods to convert Steroid intermediates to their 11 alpha 0159. Additional organisms that may be expected to hydroxy counterparts. show activity for the 11C. hydroxylation include Cepha losporium aphidicola (Phytochemistry (1996), 42(2), 411 O155 The present invention also includes the DNA 415), Cochliobolus lunatas (J. Biotechnol. (1995), 42(2), Sequences which code for the 11 alpha hydroxylases and 145-150), Tieghemella orchidis (Khim.-Farm. Zh. (1986), oxidoreductases, DNA sequences which are Substantially 20(7), 871-876), Tieghemella hyalospora Khim.-Farm. Zh. Similar and perform Substantially the same function, and (1986), 20(7), 871-876), Monosporium olivaceum (Acta DNA sequences which differ from the DNAS encoding the Microbiol. Pol, Ser. B. (1973), 5(2), 103-110), Aspergillus hydroxylases and oxidoreductases of the invention only due ustus (Acta Microbiol. Pol, Ser. B. (1973), 5(2), 103-110), to the degeneracy of the genetic code. Also included in the Fusarium graminearum (Acta Microbiol. Pol, Ser. B. present invention are the oligonucleotide intermediates used to construct mutated versions of these DNAS and the (1973), 5(2), 103-110), Verticillium glaucum (Acta Micro polypeptides encoded by these oligonucleotides and mutant biol. Pol, Ser. B. (1973), 5(2), 103-110), and Rhizopus DNAS. nigricans (J. Steroid Biochem. (1987), 28(2), 197-201). 0160 FIG. 1 sets forth the nucleotide and protein 0156 The present invention also includes antibodies Sequence of Aspergillus OchraceuS 11 alpha hydroxylase which bind specifically to A. Ochraceus 11 alpha hydroxy (SEQ ID NO: 1, SEQ ID NO: 2, respectively). FIG. 2 sets lase or A. Ochraceus oxidoreductase, including anti-peptide forth the nucleotide and protein Sequence of human oxi antibodies, methods of using these anti-peptide antibodies to doreductase (SEQ ID NO:3, SEQ ID NO: 4, respectively). purify these and other related polypeptides, methods of FIG. 3 sets forth the nucleotide and protein sequence of using the purified polypeptides to generate polyclonal or Aspergillus Ochraceus oxidoreductase (SEQ ID NO. 5, SEQ monoclonal antibodies to the full-length polypeptides, and ID NO: 6, respectively). methods of using antibodies to the full-length polypeptides to assess the presence of the polypeptides in recombinant 0.161 FIG. 4 sets forth an amino acid homology align and non-recombinant host cells. The antibodies can be used ment of A. OchraceuS 11 alpha hydroxylase cloned in to identify related polypeptides in any of a variety of host pMON45624 and aligned with related enzymes found in GenBank using BLAST FIG. 5 is a phylogenetic tree organisms that possess the biological activities associated showing the this relationship graphically. FIG. 6 shows the with these polypeptides. percent homology between Aspergillus OchraceuS Steroid 11 O157 Among the preferred organisms that can be used in alpha hydroxylase and the top 10 enzymes found in Gen this hydroxylation step are Aspergillus Ochraceus NRRL Bank using BLAST, calculated using Clustal W and Box 405, Aspergillus Ochraceus ATCC 18500, Aspergillus niger shade. ATCC 16888 and ATCC 26693, Aspergillus nidulans ATCC 0162 FIG. 7 sets forth the amino acid homology of 11267, Rhizopus Oryzae ATCC 11145, Rhizopus Stolonifer Aspergillus OchraceuS and human oxidoreductase to ATCC 6227b, Streptomyces fradiae ATCC 10745, Bacillus NADPH cytochrome P450 reductases from A. niger, mouse, megaterium ATCC 14945, Pseudomonas cruciviae ATCC and S. cerevisiae (yeast). FIG. 8 sets forth the amino acid 13262, and Trichothecium roseum ATCC 12543. Other alignment for A. Ochraceus, A. niger, and S. cerevisiae preferred organisms include Fusarium Oxysporum f. sp. oxidoreductases. FIG. 9 is a phylogenetic tree showing the cepae ATCC 11171 and Rhizopus arrhizus ATCC 11145. relatedness of Aspergillus OchraceuS and human oxi 0158 Other organisms that have exhibited activity for doreductase to reductases from A. niger, yeast, and mouse. this reaction include Absidia coerula ATCC 6647, Absidia FIG. 10 shows the percent homology between Aspergillus glauca ATCC 22752, Actinomucor elegans ATCC 6476, OchraceuS Steroid 11 alpha hydroxylase and the oxidoreduc Aspergillus flavipes ATCC 1030, Aspergillus fumigatus tases from A. niger, yeast, and mouse, calculated using ATCC 26934, Beauveria bassiana ATCC 7159 and ATCC Clustal W and Boxshade. US 2005/0003473 A1 Jan. 6, 2005
0163 FIG. 11-Alignment of human oxidoreductase transformed organism or host cell line is grown and the with top 4 hits from SwissProt. FIG. 12 sets forth a recombinant protein isolated by Standard techniques. phylogenetic tree displaying the genetic relatedness of Recombinant fusion proteins have all or a portion of a first human oxidoreductase, to these hits. FIG. 13 shows the protein joined by a linker region to a all or a portion of percent identity between human oxidoreductase and top 4 Second protein. hits from SwissProt. 0172 Hybridization 0164 FIG. 14 sets forth an immunoblot illustrating expression of Aspergillus Ochraceus P450 11 alpha hydroxy 0173 Nucleic acid molecules and fragment nucleic acid lase in baculovirus-infected insect cells harvested at 25 and molecules encoding 11 alpha hydroxylases or oxidoreduc 48 hours post infection. The nitrocellulose membrane was tases can specifically hybridize with other nucleic acid probed with a 1:1 mixture of antibodies prepared from two molecules. Two nucleic acid molecules are Said to be rabbits immunized with a conjugated Synthetic peptide, capable of Specifically hybridizing to one another if the two 11a(OH peptide 2 (SEQ ID NO 24). molecules are capable of forming an anti-parallel, double Stranded nucleic acid structure. A nucleic acid molecule is 0165 FIG. 15 sets forth an immunoblot illustrating said to be the “complement” of another nucleic acid mol expression of Aspergillus ochraceus P450 oxidoreductase in ecule, if they exhibit complete complementarity. Molecules baculovirus-infected insect cells harvested at 25 and 48 exhibit “complete complementarity” when every nucleotide hours post infection. The nitrocellulose membrane was of one of the molecules is complementary to a nucleotide of probed with a 1:1 mixture of antibodies prepared two rabbits the other. Two molecules are “minimally complementary” if immunized with a conjugated Synthetic peptide, OXr peptide they can hybridize to one another with sufficient stability to 1 (SEQ ID NO 26). permit them to remain annealed to one another under at least 0166 FIG. 16 sets forth an HPLC tracing illustrating the conventional "low-Stringency' conditions. Similarly, the conversion of androstenedione (AD) to its 11 alpha hydroxy molecules are “complementary’ if they can hybridize to one counterpart after incubating AD with Subcellular fractions another with Sufficient stability to permit them to remain prepared from baculovirus-infected insect cells expressing annealed to one another under conventional “high-Strin Aspergillus OchraceuS 1 1 alpha hydroxylase and human gency' conditions. Conventional Stringency conditions are oxidoreductase. described by Sambrook, et al., Molecular Cloning, A Labo ratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold 0167 Cloning Techniques Spring Harbor, N.Y. (1989), and by Haymes, et al. Nucleic 01.68 Genetic engineering techniques now Standard in Acid Hybridization, A Practical Approach, IRL Press, Wash the art (U.S. Pat. No. 4,935,233 and Sambrook et al., ington, D.C., 1985). Departures from complete complemen “Molecular Cloning A Laboratory Manual”, Cold Spring tarity are therefore permissible, as long as Such departures Harbor Laboratory, 1989) may be used in the construction of do not completely preclude the capacity of the molecules to the DNA sequences of the present invention. One such form a double-Stranded Structure. method is cassette mutagenesis (Wells et al., Gene 34:315 0.174 Appropriate stringency conditions which promote 323, 1985) in which a portion of the coding sequence in a DNA hybridization are well known to those skilled in the art, plasmid is replaced with Synthetic oligonucleotides that or can be found in Current Protocols in Molecular Biology, encode the desired amino acid Substitutions in a portion of John Wiley & Sons, N.Y., 6.3.1-6.3.6., (1989). Basic condi the gene between two restriction sites. tions would include, for example, 6x Sodium Saline citrate 0169 Pairs of complementary synthetic oligonucleotides (SSC) at about 45° C., followed by a wash of 2XSSC at 50° encoding the desired gene can be made and annealed to each C. Stringency can be varied, for example, by altering the Salt other. The DNA sequence of the oligonucleotide would concentration in the wash step from about 2xSSC at 50 C. encode Sequence for amino acids of desired gene with the (moderately low stringency) to about 0.2xSSC at 50° C. exception of those substituted and/or deleted from the (high Stringency). Stringency can also be altered by chang Sequence. ing the temperature in the wash Step, from room tempera ture, about 22°C. (low stringency conditions), to about 65 0170 Plasmid DNA can be treated with the chosen C. (high Stringency conditions). Both temperature and Salt restriction endonucleases then ligated to the annealed oli may be varied, or either the temperature or the Salt concen gonucleotides. The ligated mixtures can be used to transform tration may be held constant while the other variable is competent E. coli cells which will confer resistance to an changed. appropriate antibiotic. Single colonies can be picked and the plasmid DNA examined by restriction analysis or by DNA 0175 Expression Vectors Sequencing to identify plasmids with the desired genes. 0176 Another aspect of the present invention includes 0171 Cloning of DNA sequences encoding novel pro plasmid DNA vectors for use in the expression of these teins and fusion proteins may be accomplished by the use of novel hydroxylases and oxidoreductases. These vectors con intermediate vectors. Linkers and adapters can be used to tain the novel DNA sequences described above which code join DNA sequences, and to replace lost Sequences, where a for the novel polypeptides of the invention. Appropriate restriction site is internal to the region of interest. DNA vectors which can transform microorganisms or cell lines encoding a single polypeptide or a fusion protein (compris capable of expressing the hydroxylases and oxidoreductases ing a first polypeptide, a peptide linker, and a Second include expression vectors comprising nucleotide Sequences polypeptide) is inserted into a Suitable expression vector coding for the hydroxylases and oxidoreductases joined to which is then transformed or transfected into appropriate transcriptional and translational regulatory Sequences which bacterial, fungal, insect, or mammalian host cells. The are Selected according to the host cells used. US 2005/0003473 A1 Jan. 6, 2005
0177 Vectors incorporating modified sequences as 0182 Native and engineered yeast promoters suitable for described above are included in the present invention and use in the present invention have been reviewed by are useful in the production of the hydroxylases and oxi Romanos et al., Yeast 8:423-488 (1992). Most preferably, the doreductases. The vector employed in the method also protein or fragment thereof of the present invention is contains Selected regulatory Sequences in operative associa secreted by the yeast cell (Blobel and Dobberstein, J. Cell tion with the DNA coding sequences of the invention and Biol. 67:835-851 (1975); Kurjan and Herskowitz, Cell which are capable of directing the replication and expression 30:933-943 (1982); Bostian et al., Cell 36:741-751 (1984); thereof in selected host cells. Rothman and Orci, Nature 355:409–415 (1992); Julius et al., 0.178 Methods for producing the hydroxylases and oxi Cell 32:839-852 (1983); and Julius et al., Cell 36:309-318 doreductases is another aspect of the present invention. The (1984)). method of the present invention involves culturing Suitable 0183 Mammalian cells or cell lines, which has been transformed with a vector containing a DNA sequence encoding novel hydroxylases 0.184 General methods for expression of foreign genes in and oxidoreductases. Suitable cells or cell lines may be mammalian cells have been reviewed (Kaufman, R.J., 1987, bacterial cells. For example, various Strains of E. coli are “Genetic Engineering, Principles and Methods”, Vol. 9, J. K. well-known as host cells in the field of biotechnology. Setlow, editor, Plenum Press, New York; Colosimo et al., Examples of such strains include E. coli strains DH5 alpha, Biotechniques 29:314-331, 2000). Recombinant proteins are DH10B and MON105 (Obukowicz et al., Applied Environ generally targeted to their natural locations within the host cell (e.g., cytoplasm, nucleus, or various membrane com mental Microbiology 58:1511-1523, 1992). Also included in partments), or are Secreted, if a signal peptide is present. An the present invention is the expression of the hydroxylases expression vector is constructed in which a strong promoter and oxidoreductases utilizing a chromosomal expression capable of functioning in mammalian cells drives transcrip vector for E. coli based on the bacteriophage Mu (Weinberg tion of a eukaryotic Secretion signal peptide coding region, et al., Gene 126:25-33, 1993). Various other strains of which is translationally joined to the coding region for the bacteria, including the Enteric bacteria (e.g., Salmonella sp.) desired protein. For example, plasmids Such as pcDNA and B. Subtilis, may also be employed in this method. I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, Calif.) can be used. The eukaryotic 0179 When expressed in the E. coli cytoplasm, the gene Secretion signal peptide coding region can be from the gene encoding the proteins of the present invention may also be itself or it can be from another Secreted mammalian protein constructed Such that at the 5' end of the gene codons are (Bayne, M. L. et al., Proc. Natl. Acad. Sci. USA 84:2638 added to encode Met°-Ala, Met°-Ser', Met°-Cys, or 2642, 1987). After construction of the vector containing the Met' at the N-terminus of the protein. The N termini of gene, the Vector DNA is transfected into mammalian cells proteins made in the cytoplasm of E. coli are affected by such as the COS7, HeLa, BHK, Chinese hamster ovary post-translational processing by methionine aminopeptidase (CHO), or mouse L lines. The cells can be cultured, for (Ben Bassat et al., J. Bacteriol. 169:751-757, 1987), and example, in DMEM media (JRH Scientific). The polypep possibly by other peptidases, So that upon expression the tide Secreted into the media can be recovered by Standard methionine is cleaved off the N-terminus. The proteins of the biochemical approaches following transient expression for present invention may include polypeptides having Met, 24-72 hours after transfection of the cells or after establish Ala', Ser', Cys, Met-Ala, Met-Ser', or Met ment of stable cell lines following selection for antibiotic Cys' at the N-terminus. These mutant proteins may also be resistance. The Selection of Suitable mammalian host cells expressed in E. coli by fusing a Secretion signal peptide to and methods for transformation, culture, amplification, the N-terminus. This signal peptide is cleaved from the Screening and product production and purification are polypeptide as part of the Secretion process. known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625, 1981, or alternatively, Kaufman et al, Mol. 0180 Yeast Cell. Biol., 5(7): 1750-1759, 1985) or Howley et al., and U.S. Pat. No. 4,419,446. Other Suitable mammalian cell lines are 0181 Many strains of yeast cells known to those skilled the monkey COS-1 cell line and the CV-1 cell line. in the art are also available as host cells for expression of the polypeptides of the present invention. Under another 0185. Mammalian cells can also be used to express the embodiment, the protein or fragment thereof of the present nucleic acid molecules of the present invention. The nucleic invention is expressed in a yeast cell, preferably Saccharo acid molecules of the present invention can be cloned into a myces cerevisiae. The proteins or fragments thereof of the Suitable retroviral vector (See, e.g., Dunbar et al., Blood present invention can be expressed in S. cerevisiae by fusing 85:3048-3057 (1995); Baum et al., J. Hematother. 5:323-329 it to the N-terminus of the URA3, CYC1 or ARG3 genes (1996); Bregni et al., Blood 80: 1418-1422 (1992); Boris (Guarente and Ptashne, Proc. Natl. Acad. Sci. (U.S.A.) Lawrie and Temin, Curr. Opin. Genet. Dev. 3:102-109 78:2199-2203 (1981); Rose et al., Proc. Natl. Acad. Sci. (1993); Boris-Lawrie and Temin, Annal. New York Acad. (U.S.A.) 78:2460-2464 (1981); and Crabeel et al., EMBO J. Sci. 716:59-71 (1994); Miller, Current Top. Microbiol. 2:205-212 (1983)). Alternatively, proteins or fragments Immunol. 158:1-24 (1992)), adenovirus vector (Berkner, thereof of the present invention can be fused to either the BioTechniques 6:616-629 (1988); Berkner, Current Top. PGK or TRP1 genes (Tuite et al., EMBO J. 1:603-608 Microbiol. Immunol. 158:39-66 (1992); Brody and Crystal, (1982); and Dobson et al., Nucleic Acids. Res. 11:2287-2302 Annal. New York Acad. Sci. 716:90-103 (1994); Baldwin et (1983)). More preferably, the protein or fragment thereof of al., Gene Ther. 4:1142-1149 (1997)), RSV, MuSV, SSV, the present invention is expressed as a mature protein MuLV (Baum et al., J. Hematother 5: 323-329 (1996)), (Hitzeman et al., Nature 293:717-722 (1981); Valenzuela et AAV (Chen et al., Gene Ther. 5:50-58 (1998); Hallek et al., al., Nature 298:347-350 (1982); and Derynck et al., Nucleic Cytokines Mol. Ther. 2:69-79 (1996)), AEV, AMV, or CMV Acids Res. 11:1819-1837 (1983)). (Griffiths et al., Biochem. J. 241:313-324 (1987)). US 2005/0003473 A1 Jan. 6, 2005
0186 Transformation and Transfection (O'Reilly, D. R., L. K. Miller et al. Baculovirus Expression 0187. In another aspect, the invention provides a trans Vectors: A Laboratory Manual. New York, W.H. Freeman formed cell having a nucleic acid molecule which comprises and Company, 1992; and King, L.A. and R. D. Possee, The an exogenous promoter region which functions in a cell to Baculovirus Expression System. A Laboratory Guide, Lon cause the production of an mRNA molecule which is linked don, Chapman & Hall). to a structural nucleic acid molecule, wherein the Structural 0194 Abaculovirus expression vector can be constructed nucleic acid molecule encodes an 11 alpha hydroxylase or by inserting the desired gene (e.g., 11 alpha hydroxylase or oxidoreductase gene or fragment thereof. This nucleic acid oxidoreductase) into abaculovirus transfer vector which can molecule is linked to a 3' non-translated Sequence that recombine into the baculovirus genome by homologous functions in a cell to cause termination of transcription and recombination. Many transfer vectors use a Strong baculovi addition of polyadenylated ribonucleotides to a 3' end of the rus promoter (Such as the polyhedrin promoter) to drive mRNA molecule. transcription of the desired gene. Some vectors permit the expression of fusion proteins or direct the Secretion of 0188 Methods and compositions for transforming proteins from the cell by fusing a eukaryotic Secretion signal eukaryotic cells, bacteria and other microorganisms are peptide coding region to the coding region of the desired known in the art (see, for example, Sambrook et al., Molecu gene. The plasmid pVL1393 (obtained from Invitrogen lar Cloning. A Laboratory Manual, Second Edition, Cold Corp., San Diego, Calif.) can be used, for example, to direct Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., transcription of nonfused foreign genes in baculovirus (1989); Colosimo et al., Biotechniques 29:314-331, 2000). infected insect cells. The baculovirus transfer vector con 0189 Technology for introduction of DNA into cells is taining the desired gene is transfected into Spodoptera well known to those of skill in the art. Four general methods frugiperda (Sf9) insect cells along with circular or linearized for delivering a gene into cells have been described: (1) genomic baculovirus DNA, and recombinant baculoviruses chemical methods (Graham and van der Eb, Virology purified and amplified after one or more plaque assayS. 54:536-539 (1973)); (2) physical methods such as microin 0.195 Recombinant baculoviruses can also be created jection (Capecchi, Cell 22:479-488 (1980)), electroporation using the baculovirus shuttle vector System (Luckow, V. A. (Wong and Neumann, Biochem. BiophyS. Res. Commun. et al., J. Virol. 67(8):4566-4579, 1993; U.S. Pat. No. 5,348, 107:584-587 (1982); Fromm et al., Proc. Natl. Acad. Sci. 886) now marketed as the Bac-To-Bac TM Expression System (U.S.A.) 82:5824-5828 (1985); U.S. Pat. No. 5,384.253); (Life Technologies, Inc., Rockville, Md.). The desired genes and the gene gun (Johnston and Tang, Methods Cell Biol. are inserted downstream from the polyhedrin promoter in 43:353–365 (1994); (3) viral vectors (Clapp, Clin. Perinatol. mini-Tn7 cassettes that are transposed in Vivo into a bacu 20:155-168 (1993); Lu et al., J. Exp. Med 178:2089-2096 lovirus shuttle Vector genome propagated in E. coli. Com (1993); Eglitis and Anderson, Biotechniques, 6:608-614 posite viral DNAS are isolated from E. coli and transfected (1988)); and (4) receptor-mediated mechanisms (Curiel et into Sf9 cells and stocks of recombinant baculoviruses are al., Hum. Gen. Ther. 3:147-154 (1992), Wagner et al., Proc. rapidly prepared without the need for multiple rounds of Natl. Acad. Sci. (U.S.A.) 89:6099-6103 (1992)). Other tedious plaque purification common to methods that rely on methods well known in the art can also be used. homologous recombination. 0190. Transformation can be achieved using methods based on calcium phosphate precipitation, polyethylene gly 0.196 Recombinant baculoviruses can also created using col treatment, electroporation, and combinations of these the Gateway Recombinational Cloning System (Life Tech treatments (see for example Potrykus et al., Mol. Gen. nologies) of Shuttling genes from vector to vector using Genet. 205:193-200 (1986); Lorz et al., Mol. Gen. Genet. modified genetic elements (attachment sites) and modified 199: 178 (1985); Fromm et al., Nature 319:791 (1986); proteins (e.g., int, IHF, Xis) that are involved in the site Uchimiya et al., Mol. Gen. Genet. 204:204 (1986); Marcotte Specific integration and excision of bacteriophage lambda. et al., Nature 335:454-457 (1988)). 0197) Pure recombinant baculoviruses carrying the 11 0191 Assays for gene expression based on the transient alpha hydroxylase or oxidoreductase gene are used to infect expression of cloned nucleic acid constructs have been cells cultured, for example, in Excell 401 Serum-free developed by introducing the nucleic acid molecules into medium (JRH Biosciences, Lenexa, Kans) or Sf900-II (Life cells by polyethylene glycol treatment, electroporation, or Technologies). Hydroxylases or oxidoreductases that are particle bombardment (Marcotte et al., Nature 335:454-457 localized to membranes can be prepared using Standard (1988); McCarty et al., Cell 66:895-905 (1991); Hattori et protocols that fractionate and enrich for enzymes in mito al., Genes Dev. 6:609-618 (1992); Goff et al., EMBO J. chondrial or microsomal fractions (Engel and White, Dev 9:2517-2522 (1990)). Transient expression systems may be Biol. 140:196-208, 1990). Hydroxylases or oxidoreductases used to functionally dissect the regulatory and Structural that are Secreted or leak into the medium can also be features of expression cassettes comprising operably-linked recovered by Standard biochemical approaches. genetic elements. 0198 Simultaneous expression of two or more recombi nant proteins in baculovirus-infected insect cells can be 0192) Insect Cell Expression carried out by two general approaches. The Simplest 0193 Insect cells may be used as host cells to express approach is to coinfect insect cells with titered Stocks of recombinant proteins of the present invention (See, e.g., recombinant baculoviruses harboring a single heterologous Luckow, V.A., Protein Eng. J. L. Cleland., Wiley-Liss, New gene under the control of a Strong baculovirus promoter, York, N.Y.: 183-218, 1996, and references cited therein). Such as the polyhedrin or the p10 promoter. These promoters General methods for expression of foreign genes in insect are highly transcribed during the late Stages of infection cells using baculovirus vectors have been described when most host cell protein Synthesis has been shut down. US 2005/0003473 A1 Jan. 6, 2005 18
Earlier baculovirus promoters or other insect or eukaryotic Harbor Laboratory, 1989, and references cited therein, incor cell promoters can also be used to direct Synthesis at other porated herein by reference. General features and maps of a times, which generally result in lower expression levels. wide variety of cloning and expression vectors have been Varying the ratio of two or more recombinant viruses used also been published (Gacesa, P. and Ramji, D. P., Vectors: in a coinfection or Selecting viruses that use different pro Essential Data, John Wiley & Sons, 1994). General methods moters to drive expression of the recombinant protein will for the cloning and expression of genes in mammalian cells permit one skilled in the art to Select conditions Suitable for are also found in Colosimo et al., Biotechniques 29:314-331, optimal expression of the desired recombinant proteins. 2000. General and specific conditions and procedures for the 0199 Construction of dual- or multiple-expression vec construction, manipulation and isolation of polyclonal and tors will also permit the expression of two or more recom monoclonal antibodies are well known in the art (See, for binant proteins in baculovirus-infected insect cells. Gener example, Harlow and Lane, Antibodies. A Laboratory ally, these vectors permit the introduction two or more gene Manual, Cold Spring Harbor Press, Cold Spring Harbor, cassettes into a Single locus in the baculovirus genome. The N.Y., 1988). Structures of a variety of dual expression vectors have been described (O'Reilly, D. R., L. K. Miller et al. Baculovirus 0203 Unless noted otherwise, all specialty chemicals Expression Vectors: A Laboratory Manual. New York, W.H. were obtained from Sigma (St. Louis, Mo.). Restriction Freeman and Company, 1992; and King, L. A. and R. D. endonucleases and T4 DNA ligase were obtained from Life Possee, The Baculovirus Expression System: A Laboratory Technologies (Rockville, Md.), New England Biolabs (Bev Guide, London, Chapman & Hall). erly, Mass.), Roche Molecular Biochemicals (Indianapolis, Ind.), or Promega (Madison, Wis.). All parts are by weight 0200 Materials and Methods and temperatures are in degrees centigrade ( C.), unless 0201 General Methods otherwise indicated. 0202 General methods of cloning, expressing, and char 0204 Strains, Plasmids, and Sequence Cross Listings acterizing proteins are found in T. Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Labo 0205 The bacterial strains used in these studies are listed ratory, 1982, and references cited therein, incorporated in Table 1. Plasmids used or constructed for this study are herein by reference; and in J. Sambrook, et al., Molecular listed in Table 2. Brief descriptions of sequences of relevant Cloning, A Laboratory Manual, 2" edition, Cold Spring oligonucleotides, genes, or proteins are listed in Table 3.
TABLE 1.
Strains Designation Description or Genotype Reference/Source DH5C TM F, phi80 dlaczdeltaM15, Life Technologies, Rockville, delta(laczYA-argF)U169, deoR, recA1, Maryland endA1, hsdR17 (rk, mk"), phoA, supE44, lambda-, thi-1, gyrA96, relA1 DH1OBTM F, merAD(mirr-hsdRMS-merBC) Life Technologies, Rockville, phi&OdlaczDM15 DlacX74 endA1 Maryland recA1 deoR D(ara, leu)7697 araD139 galu galK nupG rpsL DH1OBac TM DH1 OB harboring the baculovirus Life Technologies, Rockville, shuttle vector bMON14272 (Kan.) Maryland; See also Luckow et al., J. Virol. and the helper plasmid pMON7124 67: 4566-4579 (1993) (Tetr)
0206
TABLE 2
Plasmids
SEO ID Plasmid NO. Marker Description Source pFastBac1 Amp Baculovirus donor plasmid Life Gent containing multiple cloning Technologies site downstream of an Inc. (Rockville, AcNPV polyhedrin MD); See also promoter within a mini-TnT Luckow et al., transposable element J. Vro. 67: capable of being transposed 4566-4579 to a baculovirus shuttle (1993) wector US 2005/0003473 A1 Jan. 6, 2005 19
TABLE 2-continued
Plasmids
SEO ID Plasmid NO. Marker Description Source pBluescript II Amp Multifunctional phagemid Stratagene, La SK cloning vector derived from Jolla, CA pUC19. pCRII-TOPO Multifunctional cloning Invitrogen, vector for direct cloning of Carlsbad, CA polymerase chain reaction products using the T Overhang pSport1 Multifunctional cloning Life vector for cloning and in Technologies, vitro transcription from Rockville, MD either strand using SP6 or T7 promoters pGEM-T A derivative of pGEM-5Zf(+) Promega, with single 5' T overhangs Madison, WI at the insertion site to improve the efficiency of PCR product ligation pMON45624 pFastEac1 EcoRIIXbal + PCR This work Gent. ragment EcoRI/XbaI encoding Aspergillus Ochraceus 11 alpha hydroxylase MON4 5603 pBluescriptII SK This work BamHI/HincII+ BamHI/HincII 5" segment of human oxidoreductase MON4 5604 Ampt pBluescriptII SK This work HincII/KpnI + HincII/KpnI 3' segment of human Oxidoreductase MON4 5605 #3 pFastBac1 BamHI/KpnI + BamHI/KpnI This work Gent. complete coding region of human oxidoreductase cDNA. MON4 Ampt pCRII-TOPO Salf BamHI + Salf BamHI This work Kan. 5" segment of A. Ochraceus Oxidoreductase cDNA MON4 5631 pCRII-TOPO BamHI/XhoI + BamHI/XhoI This work Kan. 3' segment of A. Ochraceus Oxidoreductase cDNA which lacked the intron. pMON45632 Amp pFastBac1 SalI/XhoI + containing This work Gent. assembled coding region of Aspergillus Ochraceus Oxidoreductase
0207
TABLE 3
Table of Sequences
SEQ ID NO Description Length/Sequence Type
(SEQ ID NO : 01) Nucleotide sequence of 1776 DNA Aspergillus ochraceus 11alphaOH gene from plMON45624
(SEQ ID NO : 02) Aspergillus ochraceus 514 Protein 11alphaoH protein sequence from plMON45624 US 2005/0003473 A1 Jan. 6, 2005
TABLE 3-continued Table of Sequences SEQ ID NO Description Length/Sequence Type
(SEQ ID NO : O3) Nucleotide sequence of human 2031 DNA oxidoreductase gene from pMON45 605 (SEQ ID NO : 04) Human oxidoreductase protein 677 Protein sequence from plMON45605
(SEQ ID NO : Nucleotide sequence of 2322 DNA Aspergillus ochraceus oxidoreductase gene from pMON45632
(SEQ ID NO : Aspergillus ochraceus 705 Protein oxidoreductase protein sequence from plMON45632 gatcggat.ccaatATGG DNA
(SEQ ID NO : Primer H. oxred 1A GAGACTCCCACGTGGAC DNA AC
(SEQ ID NO : 08) Primer H. oxred 1B CAGCTGGTTGACGAGAG DNA CAGAG
(SEQ ID NO : 09) Primer H. oxred 2A CTCTGCTCTCGTCAACC DNA AGCTG
(SEQ ID NO : O) Primer H. oxred 2B gatcgg taccttaGCTC DNA CACACGTCCAGGGAGTA G
(SEQ ID NO : 1) Primer A. oxred-for 1 GACGGIGCIGGTACAAT DNA GGA
(SEQ ID NO : 2) Primer A. oxred-rev1. TTAIGACCAIACATCIT DNA CCTGGTAGC
(SEQ ID NO : 3) Primer pSport-for 1 CAAGCTCTAATACGACT DNA CACTATAGGGA
(SEQ ID NO : 4) Primer A. oxred-rev2 CAGGAACCGATCGACCT DNA CGGAA
(SEQ ID NO : 5) Primer A. oxred-rev3 GTCACCCT CACCAGCAG DNA AGCCAATG
(SEQ ID NO : 6) Primer A. oxred-rev4 CCACATTGCGAACCATA DNA GCGTTGTAGTG
(SEQ ID NO : 7) Primer pSport-for 2 GCCAAGCTCTAATACGA DNA CTCACTATAGGGAAAGC
(SEQ ID NO : 8) Primer A. oxred-for 2 gtogacATGGCGCAACT DNA CGATACTCTC
(SEQ ID NO : 9) Primer A. oxred-rev5 citcgagttaGGACCAGA DNA CATCGTCCTGGTAG
(SEQ ID NO : 20) Primer A. oxred-for-3 GGATCCCTCGCGACCTG DNA TGATCA
(SEQ ID NO : 21 ) Primer A. oxred-for 4 CGAAGATTTCTTGTACA DNA AGGATGAATGGAAGACT TTTC
(SEQ ID NO : 22) Primer A. oxred-rev6 CTGAAAAGTCTTCCATT DNA CATCCTTGTACAAGAAA TC
(SEQ ID NO : 23) 11aOH peptide 1 AAAYWLATLQPSDLPEL Protein N US 2005/0003473 A1 Jan. 6, 2005 21
TABLE 3-continued Table of Sequences SEQ ID NO Description Length/Sequence Type
(SEQ ID NO : 24) 11aOH peptide 2 CRQILTPYIHKRKSLKG Protein TTDE
(SEQ ID NO : 25) 11aoH peptide 3 HMGFGHGWHACPGRFFA Protein SNEI
(SEQ ID NO : 26) oxir peptide 1 CTYWAWAKDPYASAGPA Protein MNG
(SEQ ID NO : 27) CAA75565; cytochrome P450 Protein monooxygenase Gibberella fujikuroi (SEQ ID NO : 28) CAB91316; probable cytochrome Protein P450 monooxygenase (lova) Neurospora Crassa (SEQ ID NO : 29) CAB56503; cytochrome P450 Protein Catharanthus roseus (SEQ ID NO : 30) AAB94588; CYP71D10p Glycine Protein max (SEQ ID NO : 31) CAA75566; cytochrome P450 Protein monooxygenase Gibberella fujikuroi (SEQ ID NO : 32) AAD34552; cytochrome P450 Protein monooxygenase Aspergillus terreus (SEQ ID NO : 33) CAA75567; cytochrome P450 Protein monooxygenase Gibberella fujikuroi (SEQ ID NO : 34) CAA76703; cytochrome P450 Protein Gibberella fujikurol (SEQ ID NO : 35) CAA57874; unnamed protein Protein product Fusarium oxysporum (SEQ ID NO : 36) CAA9 1268; similar to Protein cytochrome P450-cDNA EST yk423b11.3 comes from this gene; Caenorhabditis elegans (SEQ ID NO : 37) BAA02936 NADPH-cytochrome P450 Protein reductase precursor Saccharomyces cerevisiae (SEQ ID NO : 38) CAA81550 NADPH cytochrome P450 Protein oxidoreductase Aspergillus niger
(SEQ ID NO : 39) BAA04496 NADPH-cytochrome P450 Protein oxidoreductase Mus musculus
(SEQ ID NO : 40) Universal bacteriophage M13 CAG GAA ACA GCT DNA reverse primer ATG AC
(SEQ ID NO : 41) Universal bacteriophage T7 TAA TAC GAC TCA DNA promoter primer CTA TAG GG
(SEQ ID NO : 42) Aspergillus ochraceus Primer gatcgaatticATGCCCT DNA 11alphaOH- for TCTTCACTGGGCT
(SEQ ID NO : 43) Aspergillus ochraceus Primer gatctotagattacaca DNA 11alphaOH-rev gttaaactc.gc.caTATC GAT US 2005/0003473 A1 Jan. 6, 2005 22
TABLE 3-continued Table of Sequences SEQ ID NO Description Length/Sequence Type (SEQ ID NO : 44) pFastBac1 Primer Bacfwd CTGTTTCGTAACAGTT DNA TTG
(SEQ ID NO : 45) pFastBac1 Primer PolyA CCTCTACAAATGTGGTA DNA TG
(SEQ ID NO : 46) Aspergillus ochraceus Primer GAGATCAAGATTGCCTT DNA 45624-for1
(SEQ ID NO : 47) Aspergillus ochraceus Primer CTTCGACGCTCTCAA DNA 45624-for2
(SEQ ID NO : 48) Aspergillus ochraceus Primer GCAATCTTGACTCGTT DNA 45 624-rev1.
(SEQ ID NO : 49) S90 469 human cytochrome P450 2403 DNA reductase placental mRNA Partial, 2403 nt. (SEQ ID NO : 50) AAB21814 human cytochrome P450 676 Protein reductase, placental partial (SEQ ID NO : 51) A60557 human NADPH 677 Protein ferrihemoprotein reductase (SEQ ID NO : 52) PI6435 Human NADPH-cytochrome 677 Protein P450 reductase
(SEQ ID NO : 53) P00389 Rabbit NADPH-cytochrome 6.79 Protein P450 reductase
(SEQ ID NO : 54 ) P00388 Rat NADPH-cytochrome 678 Protein P450 reductase
(SEQ ID NO : 55) P37040 Mouse NADPH-cytochrome 678 Protein P450 reductase
(SEQ ID NO : 56) P04 175 Pig NADPH-cytochrome 678 Protein P450 reductase
(SEQ ID NO : 57) Universal bacteriophage SP6 gatttaggtgacactat DNA primer ag
(SEQ ID NO : 58) NotI-poly-dT adapter 5'-pgACTAGT DNA TCTAGA TCGCGA GCGGCCGC CC
(SEQ ID NO : 59) SalI adapter top strand DNA
(SEQ ID NO : 60) SalI adapter, bottom strand DNA
(SEQ ID NO : 61) Primer oxred 1C GTGGACCACAAGCTCGT DNA ACTG
(SEQ ID NO : 62) Primer oxred 2C CATCGACCACCTGTGTG DNA AGCTG
(SEQ ID NO : 63) Primer oxred 2D GTACAGGTAGTCCTCAT DNA CCGAG
(SEQ ID NO : 64) Aspergillus niger NADP CYP450 3710 DNA oxidoreductase Z26838
(SEQ ID NO : 65) Aspergillus niger NADP CYP450 693 Protein oxidoreductase CAA81550 US 2005/0003473 A1 Jan. 6, 2005 23
0208 Specific Methods 0214) DNA Sequencing Protocols 0209 Transformation of E. coli Strains 0215 Purified plasmid DNA is resuspended in dHO and its concentration is determined by measuring the absorbance 0210 E. coli strains such as DH5 alpha and DH.10B (Life at 260/280 nm in a Baush and Lomb Spectronic 601 UV Technologies, Rockville, Md.) are routinely used for trans Spectrometer. DNA Samples are Sequenced using ABI PRISMTM DyeDeoxyTM terminator sequencing chemistry formation of ligation reactions and are the hosts used to (Applied Biosystems Division of Perkin Elmer Corporation, prepare plasmid DNA for transfecting mammalian cells. E. Lincoln City, Calif.) kits (Part Number 401388 or 402078) coli strains, such as DH.10B and MON105 (Obukowicz, et according to the manufacturer's Suggested protocol. Occa al., Appl. and Envir. Micr., 58:1511-1523, 1992) can be used sionally, 5% DMSO is added to the mixture in repeat for expressing the proteins of the present invention in the experiments, to facilitate the Sequencing of difficult tem cytoplasm or periplasmic space. plates. 0211 DH10B and DH5alpha subcloning efficiency cells 0216 Sequencing reactions are performed in a DNA thermal cycler (Perkin Elmer Corporation, Norwalk, Conn.) are purchased as competent cells and are ready for trans following the recommended amplification conditions. Typi formation using the manufacturer's protocol. Other E. coli cally, DNA samples were prepared containing 500 ng of Strains are rendered competent to take up DNA using a template DNA and 100 ng of primer of choice in thin-walled CaCl, method. Typically, 20 to 50 mL of cells are grown in 0.2 mL PCR tubes that have been brought to 12 uL with LB medium (1% Bacto-tryptone, 0.5% Bacto-yeast extract, Millipore milli-Q (mCP)-quality water. 2 ul of 2 mM Mg" 150 mM NaCl) to a density of approximately 1.0 absorbance was added to each tube. Tubes were denatured for 5 minutes unit at 600 nanometers (OD600) as measured by a Baush & at 96° C. in a Perkin-Elmer System 9700 thermal cycler. Lomb Spectronic spectrophotometer (Rochester, N.Y.). The After denaturation, the tubes were chilled to a temperature cells are collected by centrifugation and resuspended in of 4° C. by the thermal cycler. 6 ul of ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit was one-fifth culture volume of CaCl solution 50 mM CaCl, added to each tube. The samples were returned to the 10 mM Tris-Cl ((10 mM 2-amino-2-(hydroxymethyl) 1,3- thermal cycler and cycle-Sequenced using the following propanediol hydrochloride, pH 7.4 and are held at 4 C. for program: (1) 96° C. for 30 sec; (2) 50° C. for 5 sec; (3) 60° 30 minutes. The cells are again collected by centrifugation C. for 4 min, followed by step (1) for 24 additional cycles and resuspended in one-tenth culture Volume of CaCl2 and then held at 4 C. Cycle sequencing was complete after Solution. Ligated DNA is added to 0.1 ml of these cells, and about 2.5 hours. the samples are held at 4 C. for 30-60 minutes. The samples 0217 Samples are purified to remove excess dye termi are shifted to 42 C. for 45 seconds and 1.0 ml of LB is nators with using Centri-SepTM spin columns (Princeton added prior to shaking the samples at 37 C. for one hour. Separations, Adelphia, N.J.) or purified through a Millipore Cells from these samples are spread on plates (LB medium MAHV N45 50 Multiscreen-HV filtration plate which had plus 1.5% Bacto-agar) containing either amplicillin (100 been filled with 25 uL Sephadex G-50 Superfine resin and micrograms/mL, ug/ml) when selecting for amplicillin-resis 300 uL mO water. Before loading samples onto filtration tant transformants, or spectinomycin (75 ug/ml) when plates, the plate was prespun in a centrifuge at 750xg for 2 Selecting for Spectinomycin-resistant transformants. The min to remove exceSS Water. The Samples were loaded onto the resin and the plate spun again at 750xg for 4 min. The plates are incubated overnight at 37 C. Colonies are picked purified Sample was collected into a 96-well plate that was and inoculated into LB plus appropriate antibiotic (100 placed directly underneath the SephadeX-filled plate during ug/ml amplicillin or 75ug/ml spectinomycin) and are grown the spin. The liquid in the 96-well plate was dried at room at 37 C. while shaking. temperature in a Speed Vac. After 45-60 min the DNA was dried and pelleted at the bottom of the plate. Samples were 0212 DNA Isolation and Characterization resuspended in 3 ull of a formamide/blue Dextran loading dye and were heated for 2 minutes (see p.33 of Perkin-Elmer 0213 Plasmid DNA can be isolated by a number of Big Dye manual for loading buffer recipe). Samples were different methods and using commercially available kits loaded onto 48 cm well-to-read length 4.5% acrylamide gels known to those skilled in the art. Plasmid DNA is isolated and Sequenced for 7 hr using ABI automated DNA sequenc using the Promega Wizard TM Miniprep kit (Madison, Wis.), ers (typically run module Seq Run 48E-1200 and dye set DT, the Qiagen QIAwell Plasmid isolation kits (Chatsworth, Program BD, Set Any-Primer). Calif.) or Qiagen Plasmid Midi or Mini kit. These kits follow the same general procedure for plasmid DNA isolation. 0218 Overlapping DNA sequence fragments are ana Briefly, cells are pelleted by centrifugation (5000xg), the lyzed and assembled into master DNA contigs using plasmid DNA released with sequential NaOH/acid treat Sequencher DNA Analysis software (Gene Codes Corpora ment, and cellular debris is removed by centrifugation tion, Ann Arbor, Mich.) or the Perkin-Elmer Data Collection (10000xg). The Supernatant (containing the plasmid DNA) and Sequence Analysis programs to assign bases to the data is loaded onto a column containing a DNA-binding resin, the collected. column is washed, and plasmid DNA eluted. After Screening 0219 BLAST, ClustalW, and Boxshade Homology for the colonies with the plasmid of interest, the E. coli cells Alignment Tools are inoculated into 50-100 ml of LB plus appropriate anti biotic for overnight growth at 37 C. in an air incubator 0220 A variety of programs can be used to align nucle while shaking. The purified plasmid DNA is used for DNA otide or peptide Sequences to each other and to facilitate Sequencing, further restriction enzyme digestion, additional homology Searches in large Sequence databases. BLAST subcloning of DNA fragments and transfection into E. coli, (Basic Local Alignment Search Tool), which implements the mammalian cells, or other cell types. statistical matching theory by Karlin and Altschul (Proc. US 2005/0003473 A1 Jan. 6, 2005 24
Natl. Acad. Sci. USA 87:2264-2268, 1990; Proc. Natl. Acad. 0225 Boxshade v 3.31 is a public domain program for Sci. USA 90:5873-5877, 1993), is a widely used program for creating nicely formatted printouts from muliple-aligned rapidly detecting ungapped nucleotide or peptide Subse protein or DNA sequences. Boxshade, by itself, does not quences that match a given query sequence (Available from create alignments, but applies shading or coloring to files the National Center for Biotechnology Information, http:// that were previously prepared by other Sequence alignment www.ncbi.nlm.nih.gov). BLAST uses a heuristic algorithm programs. The inputs to Boxshade are the alignments cre which seeks local as opposed to global alignments and is ated by ClustalW and the threashold values for the residues therefore able to detect relationships among Sequences to be colored or shaded. In most cases, except where specified, a 50% identity value was used. With this setting, which share only isolated regions of similarity (Altschulet if a position has greater than or equal to half of the Sequences al., J. Mol. Biol. 215:403-410, 1990). Sharing an identical residue, then it is shaded. Boxshade is 0221) Two parameters can be varied which alter the available by ftp from ftp. or by e-mail from Kay Hofmann sensitivity and quantity of BLAST search results. Parameter (khofmann(a)isrec-sun1-unilch or Michael D. Baron B (with a default value of 10) regulates the number of (michael.baron(abbSrc.ac.uk). high-scoring segment pairs (alignments) reported in the 0226 Protein Purification and Characterization results. Parameter V (with a default value of 10) is the 0227 Protein purification can be accomplished using any maximum number of database Sequences (hits) for which of a variety of chromatographic methods Such as: ion one-line descriptions will be reported. Matches are based on eXchange, gel filtration or hydrophobic chromatography or high-scoring segment pairs (HSPs). Two Sequences may reversed phase HPLC. In Some cases, proteins which are share more than one HSP, if the HSPs are separated by gaps. properly folded can be affinity-purified using affinity The BLAST algorithm is sensitive to ambiguities in the reagents, Such as monoclonal antibodies or receptor Subunits Sequence and is not well-Suited for Sequences that contain attached to a Suitable matrix. These and other protein many gaps. purification methods are described in detail in Methods in 0222. The program blastp compares an amino acid query Enzymology, Volume 182 “Guide to Protein Purification” Sequence against a protein Sequence database. blastin com edited by Murray Deutscher, Academic Press, San Diego, pares a nucleotide query Sequence against a nucleotide Calif., 1990. Sequence database. blastX compares a nucleotide query 0228. The purified protein can be analyzed by RP-HPLC, Sequence translated in all reading frames against a protein electrospray mass spectrometry, and SDS-PAGE. Protein Sequence database. You could use this option to find poten quantitation is done by amino acid composition, RP-HPLC, tial translation products of an unknown nucleotide Sequence. and/or Bradford protein dye-binding assays. In Some cases, tblastin compares a protein query Sequence against a nucle tryptic peptide mapping is performed in conjunction with otide Sequence database dynamically translated in all read electrospray maSS spectrometry to confirm the identity of the ing frames. thlastX compares the six-frame translations of a protein. nucleotide query Sequence against the Six-frame translations of a nucleotide Sequence database (See http://www.ncbi.n- EXAMPLES lm.nih.gov/Education/BLASTinfo/ for more information on BLAST, related programs, and pattern matching algo 0229. The following examples will illustrate the inven rithms). tion in greater detail, although it will be understood that the invention is not limited to these Specific examples. Various 0223) Nucleotides searches performed with BLAST, other examples will be apparent to the person skilled in the score=98-557, word length 514 letters, were used to obtain art after reading the present disclosure without departing nucleotide Sequences homologous to nucleic acid molecules from the Spirit and Scope of the invention. It is intended that of the present invention. Protein searches are performed with all such other examples be included within the scope of the BLASTP, score=50, word length=3 to obtain amino acid appended claims. Sequences homologous to a reference polypeptide (e.g., SEQ ID NO: 2). Example 1 0224 Clustal W version 1.74, which implements a dif ferent algorithm for alignment of multiple DNA or protein Preparation of A. Ochraceus Spores for RNA Sequences, was also used to prepare alignments and to assign Extraction percent identities between different Sequences. This program 0230. Aspergillus ochraceus ATCC 18500 stock culture improves the Sensitivity of progressive multiple Sequence (50 ul) was grown for 3-4 days on plates containing sporu alignment through Sequence Weighting, position Specific gap lation medium: 50 g/L molasses, 5 g/L cornsteep liquid, 5 penalties and weight matrix choice (Thompson et al., g/L KHPO, 25 g/L NaCl, 25 g/L glucose, 20 g/L agar, and Nucleic Acids Research, 22(22):4673-4680, 1994). The 0.4 g/L progesterone, pH 5.8. Progesterone was included in default parameters for version 1.74 were used facilitate the media to induce the Steroid 11 O-hydroxylase. Spores alignments and to assign percent identities between two were Scraped from the plates into 5 to 7 ml Saline, washed Sequences. The input consisted of Sequences in FASTA in Saline, collected by centrifugation, and Suspended in format and the output is the alignment shown in the figures. Saline containing 15% glycerol. The Spores were frozen on For nucleic acid Sequences, the iub DNA weight matrix was dry ice and stored at -80 C. Approximately 0.8 g. spores used. For amino acid Sequences, the blosum protein weight were incubated at 30° C. in a 1 liter flask containing 400 ml matrix was used (See http://www.ncbi.nlm.nih.gov/Educa 1% glucose, 50 mM. KHPO and 0.1 g canrenone, pH 7.0. tion/BLASTinfo/ for more information on BLAST, related This treatment prior to Spore disruption has three benefits: programs, and pattern matching algorithms. (1) to induce the steroid 11 C-hydroxylase by incubation US 2005/0003473 A1 Jan. 6, 2005 25 with canrenone; (2) to determine whether the spores were ered by centrifugation and washed with 70% ethanol. The catalyzing the 11 O-hydroxylation of canrenone; (3) and to RNA was resuspended in 10 ml water, re-extracted with Soften the Spore wall. After approximately 26 hours of chloroform and precipitated with ethanol overnight at -20° incubating with shaking at 30° C. to provide better aeration, C. Total RNA (3 mg) was recovered by centrifugation and the Spores were collected by centrifugation. Visual inspec rehydrated in 2 ml water, and precipitated on ice by adding tion with the aid of a microscope indicated that very few had an equal volume of cold 4 M lithium chloride. This precipi Started to germinate. The Spore pellets were flash frozen in tation was done to remove DNA, carbohydrates, heme, and liquid nitrogen and stored at -80 C. The media was other impurities which can carry over from guanidine meth analyzed for presence of 11 alpha hydroxy canrenone by ods. The RNA was recovered by a 25 minute centrifugation. HPLC to determine whether spores used for library con Example 5 Struction demonstrated the desired activity. Example 2 Extraction of Total RNA from Induced Mycelia 0234 Approximately 0.5g wet weight cells were pulver A. OchraceuS Spores Catalyze 11 O-hydroxylation ized to a fine powder under liquid nitrogen with a mortar and of Canrenone pestle pre-chilled in dry ice. The powder was added to 10 ml 0231. Approximately 160 ml of media from the spore Trizol Reagent (Life Technologies) and homogenized with a induction was extracted three times with 70 ml ethyl acetate Kinematica polytron (Kinematica AG, Lucerne, Switzer to collect the Steroid Substrate and products. The organic land) at setting #4. Cellular debris was removed by centrifu phase was dried over anhydrous magnesium Sulfate, filtered, gation prior to chloroform extraction. The aqueous phase and evaporated to dryneSS. The residue was dissolved in 8 ml containing nucleic acids was precipitated with isopropanol methanol So that the final concentration of canrenone was for 10 minutes at room temperature. The precipitate was approximately 15 mM (assuming quantitative recovery). collected by centrifugation and washed with 70% ethanol. The media extract was diluted 10- to 15-fold into 50% The RNA was rehydrated in water and re-extracted with methanol for HPLC analysis. Stock solutions of canrenone chloroform to remove any residual proteins. The aqueous and 11 O-hydroxy canrenone were prepared in methanol. phase was precipitated at -20°C. with 1/10 volume of 3 M Standards for HPLC analysis were prepared from these Sodium acetate and 2.5 volumes absolute ethanol. The final stock solutions by diluting to a final concentration of 750 uM yield was 424 ug. Approximately 4 ug and 16 ug of total with 50% methanol. Media extract and standards were RNA were separated by electrophoresis through a 1.2% chromatographed over a C-4 reverse phase HPLC column. agarose gel and Visualized by Staining in ethidium bromide. The media exhibited a component with the same retention Chromosomal DNA was present as a minor contaminant. time as the 11 O-hydroxy canrenone Standard, as monitored at 254 nm (data not shown). Example 6 Example 3 Extraction of Total RNA from HepG2 Cells Growth of A. Ochraceus Mycelia for RNA 0235 Hepatocellular human liver carcinoma cells (HepG2), ATCC HB-8065, were maintained in DMEM high Extraction glucose media Supplemented with PenStrep, glutamate and 0232 Liquid cultures of Aspergillus Ochraceus mycelia 10% fetal bovine serum (Life Technologies, Rockville, were grown in 10 g/L peptone, 10 g/L yeast extract and 10 Md.). Cells were induced overnight with 0.05% ethanol and g/L glucose containing 20 g/L canrenone for 24 to 72 hours harvested for RNA extraction by trypsinization. Briefly, the at 28° C. in a volume of 160 ml. Ten ml samples of cells cell pellet was resuspended in >10x volumes of 4 M were filtered, washed with cold water, frozen, and Stored at guanidine isothiocyanate, 50 mM Tris-HCl, pH 7.5, 25 mM -80° C. EDTA (Solution D, Life Technologies) and then vortexed. Water and sodium acetate, pH 4.1, were added such that the Example 4 final concentration of Sodium acetate was 0.1 M. The RNA Solution was extracted with one half volume of chloroform Extraction of Total RNA from Induced Spores and placed on ice for 15 minutes. The aqueous phase was 0233 Approximately 0.4 g spores were disrupted in 40 re-extracted with chloroform and precipitated overnight ml Trizol reagent (Life Technologies, Rockville, Md.) using with isopropanol. Total RNA was resuspended in solution D a Mini-BeadbeaterTM model 3110 (Biospec Products, and re-precipitated with isopropanol, followed by two pre Bartlesville, Okla.). Briefly, spore-Trizol mixture was sub cipitations in water containing 0.3 M sodium acetate pH 5.5 jected to four 30 second pulses at low speed. Between and 2.5 volumes of ethanol. PolyA selection was performed pulses, tubes containing spores were chilled on ice. Visual twice as described below. inspection with the aid of a microScope indicated that the majority of the spores were disrupted by this treatment. The Example 7 debris was pelleted by low-speed centrifugation and the total RNA in the Supernatant was extracted following the manu PolyA" Selection of mRNA facturer's recommended protocols for use with Trizol. Briefly, 2 ml chloroform was added for each 10 ml Trizol in 0236) PolyA" RNA was selected from total RNA with an 11 ml polypropylene centrifuge tubes. Following a 3 minute Eppendorf 5Prime, Inc. kit (Boulder Colo.). Briefly, each 1 extraction of proteins, phase Separation was done by cen mg of total RNA was Selected twice over a column contain trifugation and the aqueous phase containing the RNA was ing oligo dT cellulose. The column slurry was packed by transferred to a clean tube for precipitation with an equal gentle centrifugation and equilbrated with 0.5 M NaCl. RNA volume of isopropanol. The precipitated RNA was recov was allowed to bind to the dT cellulose for 15 minutes at US 2005/0003473 A1 Jan. 6, 2005 26 room temperature. The columns were washed once with 0.5 ultraViolet light next to a ruler So that the appropriate region M. NaCl, and twice with 0.1 M NaCl. PolyA RNA was of the cDNA could be recovered from the gel. eluted in 0.5 ml 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. The GENECLEAN II was used to extract the cDNA, which was selection by oligo dT cellulose was performed twice. The eluted in 20 ul water. mRNA was precipitated at -20° C. with 0.3 M sodium acetate in 50% ethanol, with glycogen added as carrier. Example 10 Example 8 Library Construction in Vector pSport1 and Electroporation into E. coli CDNA Synthesis and Library Construction 0241 An aliquot of the size-selected cDNA was ligated 0237) The SuperscriptTM Plasmid System for cDNA Syn overnight at 4 C. with pSport1 (Life Technologies, Inc., thesis and Plasmid Cloning kit (Life Technologies) was used Rockville, Md.) predigested with NotI and Sall in a 20 ul for cDNA systhesis and library construction. Superscript II reaction containing 50 mM Tris-HCl, pH 7.6, 10 mM reverse transcriptase catalyzed the first Strand of cDNA in a MgCl, 1 mM ATP, 5% (w/v) PEG 8000, 1 mM DTT, 2.5 20 ul reaction for 1 hour at 42 C. The final composition was ug/ml pSport1, approximately 0.5 ug/ml cDNA, and 50 50 mM Tris-HCl, pH 8.3, 75 mM KC1, 3 mM MgCl, 10 mM units/ml T4 DNA ligase. The ligation mixture was precipi DTT, 50 uM each dATP, dCTP, dGTP and dTTP, 50 ug/ml tated by the addition of 12.5ul 7.5 M ammonium acetate, 5 oligo-dT-Not primer-adaptors that were phosphorylated at ul yeast tRNA carrier and 70 ul absolute ethanol. The ligated their 5' end (Life Technologies) and 50,000 units/ml Super cDNA was recovered by centrifugation at room temperature Script II reverse transcriptase. for 20 minutes and rehydrated in 5 ul sterile water. One ul oligo-dT-NotI primer- adapter 5'-pCACTAGT, TCTAGA TCGCGA GCGGCCGC CC (T) 15-3' (SEQ ID NO: 58) Spel Xibal NiruI Not
0238 A radiolabeled tracer (A- PdCTP) was not of the ligated cDNA was introduced into ElectroMAX added. The second strand of cDNA was synthesized in a DH10B E. coli (Life Technologies) by electroporation. Cells reaction volume of 150 ul. The final composition of this were allowed to recover in 1 ml SOC medium (Life Tech mixture including the first strand reaction was 25 mM nologies) for 1 hour at 37 C., before plating an aliquot on Tris-HCl, pH 7.5, 100 mM KC1, 5 mM (NHA)SO, 0.15 LB with 100 ug/ml amplicillin. The titer of the Aspergillus mM B-NAD", 250 uM each dATP, dCTP, dGTP and dTTP, Ochraceus spore library (designated LIB3025) was deter 1.2 mM DTT, 65 units/ml E. coli DNA ligase, 250 units/ml mined by preparing Serial dilutions of the cell Suspension in E. coli DNA polymerase I and 13 units/ml E. coli Rnase H. SOC. The equivalent of 1 ul, 0.1 ul and 0.01 ul samples of After a 2 hour incubation at 16 C., 10 units of T4 DNA the cell Suspension were plated, and the resulting titer was polymerase was added, and incubated 5 minutes at 16 C. calculated to be 1.75x10"/ml colony forming units. The reaction was stopped with 10 ul 0.5 M EDTA and the cDNA was separated from cDNAS Smaller than 300 base Example 11 pairs, primer-adaptors and deoxynucleotides with GENECLEAN II (BIO 101 Inc. La Jolla, Calif.). Annealed Identification of Clones Encoding Cytochrome Sal I adaptors (Life Technologies) that were phosphorylated P450 Enzymes by DNA Sequence Analysis and at their 5' blunt end were ligated to the cDNA overnight at Construction of Plasmid pMON45624 Encoding 16° C. Aspergillus Ochraceus 11 Alpha Hydroxylase 0242 Cloning of 11 Alpha Hydroxylase from Aspergillus OchraceuS 5'-TCGACCCACGCGTCCG -3' (SEQ ID NO: 59) 0243 Approximately 2,000 colonies were selected on LB 3'- GGGTGCGCAGGCp-5' (SEQ ID NO: 60) agar plates containing 100 ug/ml amplicillin and miniprep plasmid DNA samples were prepared for Sequencing. Uni 0239 GENECLEAN II was used to remove the adaptors. directional Sequencing was performed from the 3' end of the The cDNA was then digested with Not. QIAquick columns expressed Sequence tags (ESTs) beginning at the Not Site (QIAGEN, Valencia, Calif.) were used to remove small encompassing part of the poly dT primer used for cDNA DNA fragments from the cDNA, which was ethanol pre Synthesis. Two universal primers were used to facilitate the cipitated. Sequencing: Example 9 M13 reverse: Size Fractionation of CDNA CAG GAA ACA GCT. ATG AC (SEQ ID NO: 40) 0240 The cDNA was enriched for species approximately T7 promoter: 1.5 kb and larger by gel electrophoresis through 0.8% TAA TAC GAC. TCA CTA TAG GG (SEQ ID NO: 41) Sea-Plaque agarose (FMC BioProducts, Rockland Me.) in TAE buffer. The preparative gel had a lane of DNA size 0244. Most known cytochrome p450s contain a con markers which was excised from the gel after electrophore Served heme-binding region approximately 50 amino acid sis and stained with ethidum bromide for visualization under residues (150 nucleotides) upstream of the stop codon US 2005/0003473 A1 Jan. 6, 2005 27
(Nelson et al., Pharmacogenetics 6:1-42, 1996). The 2,000 percent homology between Aspergillus OchraceuS Steroid 11 ESTs were Screened for Sequences encoding the canonical alpha hydroxylase and the top 10 enzymes found in Gen heme-binding motif (FXXGXXXCXG, where “X” is any Bank using BLAST, calculated using Clustal W and Box amino acid) in the appropriate region using BLASTX and shade. Visual inspection of the Sequences Scored as hydroxylases for the canonical heme-binding motif. Only fifteen ESTs had Example 12 the heme-binding motif. One EST was unique and the other fourteen appeared to be overlapping Sequences. The cDNA Amplification of cDNA Encoding Human NADPH inserts from Seven clones encoding putative cytochrome Cytochrome P450 Reductase and Cloning into p450 enzymes were then Sequenced to completion. All Seven Plasmids pMON45603, pMON45604, and encoded the same enzyme. pMON45605 0245 Gene Amplification of Aspergillus Ochraceus 11 0251 Gene Amplification of human oxidoreductase Alpha Hydroxylase 0252) Approximately 1 ug polyA mRNA from HepG2 0246 The coding region of the 11 alpha hydroxylase was cells was heated to 65 C. for 10 minutes with 100 ng amplified by PCR using a unique clone from the A. Ochra random hexamers (Invitrogen, Carlsbad, Calif.) in an 11 ul ceus cDNA spore library (LIB3025) as a template. The reaction. The mixture was chilled on ice, then incubated at primers included recognition sites for EcoRI (forward) and 42 C. for 75 minutes in a 20 ul reaction containing 1 ul Xbal (reverse) for directional cloning into pFastbac1. RNase inhibitor (Promega, Madison, Wis.), 0.01 M DTT, 5 Amplification was carried out for 32 cycles using a PCR mM dNTPs, 50 mM Tris-HCl, pH 8.3, 75 mM KC1, 3 mM core kit (Roche) and 50 pmol of each primer. One cycle MgCl, and 1 ul SuperScriptiI enzyme (Life Technologies). consisted of a denaturation step at 94 C. for 45 seconds, an The reverse transcriptase was inactivated by heating to 95 annealing step at 60° C. for 45 seconds, and an elongation C. for 2 minutes. First strand cDNA was stored at -20° C. step at 72 C. for 60 seconds. Forward and reverse primers were based on the nucleotide sequence of accession number S90469 (human placental partial mRNA encoding cytochrome P450 reductase (SEQ Primer 11alphaOH- for: ID NO: 49)). The accession number of the corresponding gatcgaattcATGCCCTTCTTCACTGGGCT (SEQ ID NO: 42) protein sequence is AAB21814 (SEQ ID NO: 50). The Primer 11alphaOH-rev: human oxidoreductase was cloned in two pieces which were gatctotagaTTACACAGTTAAACTCGCCATATC (SEQ ID NO : 43) assembled in pFastBac1 (Life Technologies) by ligation at GAT an internal HincII site. The primers included restriction sites for directional Subcloning into pFastBac1. 0247 Construction of pMON45624
0248. The amplified fragments described above were Primer H. oxred 1A: purified through a QIAquick column (Qiagen, Valencia gatcggatccaatATGGGAGACTCCCACGTGGAC (SEQ ID NO: 07) Calif.) and digested with EcoRI and Xbal prior to ligation AC into pFastBac1 cleaved with EcoRI and Xbal. The resulting Primer H. oxred 1B: plasmid was designated pMON45624 and the DNA CAGCTGGTTGACGAGAGCAGAG (SEQ ID NO : 08) Sequence verified using primers based on the Vector Sequence and internal primers based on the 11 alpha Primer H. oxred 2A: hydroxylase Sequence (shown below). CTCTGCTCTCGTCAACCAGCTG (SEQ ID NO : 09) Primer H. oxred 2B: gatcgg taccttagcTCCACACGTCCAGGGAGTA (SEQ ID NO : 10) Primer BacfWo: G CTGTTTTCGTAACAGTTTTG (SEQ ID NO: 44) Primer PolyA: 0253) The second strand was synthesized using 400 uM CCTCTACAAATGTGGTATG (SEQ ID NO: 45) dNTP and 167 nM of each primer set per 150 ul reaction. Amplification was performed with Deep Vent polymerase Primer 45 624-for 1: (New England Biolabs, Beverly, Mass.). The reaction for GAGATCAAGATTGCCTT (SEQ ID NO: 46) segment 2 (the 3' half of the oxidoreductase cDNA) was Primer 45 624-for2: adjusted to 5% DMSO. The amplification included an initial CTTCGACGCTCCAA (SEQ ID NO: 47) cycle of denaturation at 94 C. for 90 seconds, followed by Primer 45 624-revil: annealing at 62 C. for 2 minutes and elongation at 72 C. GCAATCTTGATCTCGTT (SEQ ID NO : 48) for 2 minutes. This was followed by 30 cycles, consisting of a 45 Second denaturation Step, a 45 second annealing Step, 0249. The nucleotide and predicted amino acid sequences and a 60 Second elongation Step. The elongation Step was of the cloned 11 alpha hydroxylase are displayed in FIG. 1 extended to 5 minutes for the final cycle. as SEQ ID NO: 1 and SEQ ID NO: 2, respectively. 0254 Construction of pMON45603, pMON45604, 0250 FIG. 4 sets forth an amino acid homology align pMON45605 ment of A. OchraceuS 11 alpha hydroxylase cloned in 0255. The PCR fragments for the 5' half of the oxi pMON45624 and aligned with related enzymes found in doreductase cDNA were digested with BamHI and HincII. GenBank using BLAST FIG. 5 is a phylogenetic tree The PCR fragments for the 3' half of the oxidoreductase showing the this relationship graphically. FIG. 6 shows the cDNA were digested with HincII and KpnI and ligated into US 2005/0003473 A1 Jan. 6, 2005 28 pBlueScript II (Stratagene, La Jolla, Calif.) for Sequencing. SER in A. fumigatis (CGC vs. AGC). Inosines replaced the The resulting plasmids were designated pMON45603 (5' third base in codons when there was a discrepancy between segment) and pMON45604 (3' segment). The BamHI/HincII the A. niger and A. fumigatus Sequence. fragment from pMON45603 and the HincII/KpnI fragment from pMON45604 were ligated into pFastbac1 cut with BamHI and KpnI, to generate pMON45605. Primer A. oxred-for 1: GACGGIGCIGGTACAATGGA (SEQ ID NO : 11) 0256 Sequencing primers were based on the Sequence of GenBank accession number S90469 (SEQ ID NO 49), a Primer A. oxred-revil: cDNA encoding cytochrome P450 reductase human, pla TTAIGACCAIACATCITCCTGGTAGC (SEQ ID NO: 12) centa, mRNA Partial, 2403 nt). The cognate protein (where I = Inosine) sequence is: AAB21814 (SEQ ID NO 50) cytochrome P450 reductase {EC 1.6.2.4) human, placenta, Peptide Partial, 0261) A partial cDNA clone was amplified from approxi 676 aa Homo Sapiens). The cDNA insert of pMON45603 mately 5 ug of total RNA extracted from A. Ochraceus was Sequenced using primer OXred 1C, and the cDNA insert mycelia. Before the first strand synthesis, the RNA was of pMON45604 was sequenced using primer oxred 2C and heated to 65° C. for 10 minutes with 100 ng random 2D. Universal T7 (SEQID NO: 41) and M13 reverse (SEQ hexamers (Promega Madison Wis.) in an 11 ul reaction ID NO: 40) primers, which annealed to vector sequences mixture. The mixture was chilled on ice, then incubated at flanking the cDNA inserts were also used for Sequencing. 42 C. for 75 minutes in a 20 ul reaction containing 1 ul RNase inhibitor (Promega), 0.01 M DTT, 5 mM dNTPs, 50 mM Tris-HCl, pH 8.3), 75 mM KC1, 3 mM MgCl, and 1 ul Primer oxred 1C: SuperScriptiI (LTI). The reverse transcriptase was inacti GTGCACCACAAGCTCGTACTG (SEQ ID NO : 61) vated by heating to 95 C. for 2 minutes. The first strand Primer oxred 2C: cDNA was stored at -20° C. The second strand was syn CATCGACCACCTGTGTGAGCTG (SEQ thesized using 5 ul of the first strand as template. The ID NO: 62) reaction included 500 nM primers, 200 uM each dNTP, and Primer oxred 2D : Taq polymerase and buffer as supplied in PCR core kit GTACAGGTAGTCCTCATCCGAG (SEQ (Roche Molecular Biochemicals, Indianapolis, Ind.). Ampli ID NO: 63) fication was performed using 32 cycles of a 30 Second denaturation step at 94 C., a 30 Second annealing step at 60 0257 The nucleotide and predicted amino acid sequences C. and a 60 second elongation step at 72 C. The amplified of the cloned human oxidoreductase are displayed in FIG. DNA products were cloned into pGEM-T (Promega, Madi 2 as SEQ ID NO: 3 and SEQ ID NO: 4, respectively. FIG. son, Wis.) and sequenced using universal T7 (SEQ ID NO: 11 Sets forth an alignment of human oxidoreductase with top 41) and SP6 (SEQ ID NO: 57) primers. 4 hits from SwissProt. FIG. 12 sets forth a phylogenetic tree displaying the genetic relatedness of human oxidoreductase, to these hits. FIG. 13 shows the percent identity between Primer SP6 human oxidoreductase and top 4 hits from SwissProt. GATTTAGGTGACACTATAG (SEQ ID NO: 57) Example 13 0262 Alignment of the sequences with the A. niger cprA gene revealed that the A. Ochraceus clones had an intron in Amplification of cDNA Encoding NADPH the Same position as the intron in the A. niger gene. This Cytochrome P450 Reductase from A. Ochraceus indicated that the A. Ochraceus PCR products might have and Cloning into Plasmids pMON45630, been amplified from a genomic DNA contaminant of the pMON45631, and pMON45632 total RNA. A reverse primer based on the A. Ochraceus Sequence was designed to amplify the approximately 600 0258 Gene Amplification of Aspergillus Ochraceus Oxi missing bp including the initial methionine. The A. Ochra doreductase ceus cDNA library was then used as a template for PCR. The forward primer was based on the reverse complement of 0259 An alignment of sequences from the Aspergillus vector pSport1 (Life Technologies) bases 299 to 326. The niger cprA gene accession number Z26938 (SEQ ID NO: other primer, A.OXred-rev2 was bases on the A. OchraceuS 65) and a partial cDNA clone 804561639F1 from Aspergil Sequence encoding residues 326-333. lus fumigatus (PathoSeq Database, Incyte Pharmaceuticals) was visually Scanned to Select regions of high homology for the design of primers for PCR. A primer set was selected Primer pSport-for1: which Spanned the coding region of the cprA gene product CAAGCTCTAATACGACTCACTATAGGGA (SEQ ID NO : 13) from amino acids 203 to 693. Primer A. oxred-rev 2: 0260 Primers were selected from the 5' most region of CAGGAACCGATCGACCTCGGAA (SEQ ID NO: 14) overlap where the amino acid Sequence was identical between both and the nucleic acid sequence differed by 2 0263. The A. Ochraceus spore library size made from positions in the 3" codon position. For the 3' primer, the gel-purified fragments >1.5 kb in size was then used as a nucleic acid encoding the Stop codon, last 7 amino acid template for amplifying the final 200 bases of coding region. residues and 2 additional bases corresponding to Second and Two new reverse primers were designed from the A.OXred third positions in the codon of the amino acid residue 8 Sequence, and a new forward primer based on pSport1 positions from the Stop codon encodes ARG in A. niger and (bases 295-328) was also used. US 2005/0003473 A1 Jan. 6, 2005 29
a combination of A.OXred-for3 with A.OXred-rev6, and A.oxred-for4 with A.oxred-reviš. Following a 2 minute Primer A. oxred-rev3: initial denaturation, 28 cycles of PCR amplification were GTCACCCT CACCAGCAGAGCCAATG (SEQ ID NO: 15) performed. One cycle included a 45 Second denaturation at Primer A. oxred-rev4: 94 C., a 45 second denaturation step at 62 C. and a 45 CCACATTGCGAACCATAGCGTTGTAGTG (SEQ ID NO: 16) Second elongation Step at 72 C. One ul of each reaction Primer pSport-for2: served as template for the second PCR amplification with GCCAAGCTCTAATACGACTCACTATAGGGAAAGC (SEQ ID NO : 17) primers A.OXred-for3 and A.OXred-reviš using Elongase enzyme and buffers. Amplification consisted of 30 cycles 0264 Amplification was performed using an Elongase with a 30 second denaturation step at 94 C., a 30 second polymerase kit (Life Technologies, Rockville Md) for 35 annealing step at 62 C., and a 5 minute elongation step at cycles consisting of a denaturation step at 94 C. for 30 68 C. The PCR products were directly cloned into pCRII Seconds, an annealing Step at 63 C. for 30 Seconds, and an TOPO. DNA sequencing demonstrated that the intron had elongation step at 68 C. for 5 minutes. The PCR products been removed. This clone was designated pMON45631. were cloned directly into pCRIITOPO (Invitrogen). Twelve clones were Sequenced, and the composite Sequence, 0271 Plasmid pMON45632 was constructed in a three extended for 232 bases upstream of the initial methionine, way ligation by ligating the Sall/BamHI fragment from and included 2 in-frame stop codons (Data not shown). pMON45630 with the BamHI/XhoI fragment from 0265 Primers incorporating the complete coding region pMON45631 and vector pFastBac1, which had been cut of A.oxred were designed with a 5' SalI site and a 3' XhoI with SalI and XhoI and de-phosphorylated to enhance the Site for ligation into expression vector pFastBac1. recovery of vectors with the desired inserts. 0272. The nucleotide and amino acid sequences of the Primer A. oxred-for2: cloned Aspergillus OchraceuS 11 oxidoreductase are dis gtogacATGGCGCAACTCGATACTCTC (SEQ ID NO: 18) played in FIG. 3 as SEQ ID NO: 5 and SEQ ID NO: 6, respectively. FIG. 7 sets forth the amino acid homology of Primer A. oxred-rev5: citcgagittaGGACCAGACATCGTCCTGGTAG (SEQ ID NO : 19) Aspergillus OchraceuS and human oxidoreductase to NADPH cytochrome P450 reductases from A. niger, mouse, 0266 A. Ochraceus total RNA was used as a template for and S. cerevisiae. FIG. 8 sets forth the amino acid alignment PCR with these primers and the Elongase kit. Amplification for A. Ochraceus, A. niger, and S. cerevisiae oxidoreductases. FIG. 9 is a phylogenetic tree showing the relatedness of consisted of 35 cycles with a 30 second denaturation step at Aspergillus Ochraceus and human oxidoreductase to reduc 94 C., a 30 second annealing step at 64 C., and a 5 minute tases from A. niger, yeast, and mouse. FIG. 10 shows the elongation step at 68 C. An aliquot of the cDNA from percent homology between Aspergillus OchraceuS Steroid 11 reaction ran as a single band of approximately 2.1 kb. alpha hydroxylase and the oxidoreductases from A. niger, Construction of pMON45630 yeast, and mouse, calculated using ClustalW and Boxshade. 0267. The PCR products were cloned directly into pCRII Example 15 TOPO (Invitrogen, Carlsbad, Calif.). All clones contained the internal intron noted earlier. One clone was designated Generation of Polyclonal Antibodies Recognizing pMON45630. Aspergillus Ochraceus 11 Alpha Hydroxylase and 0268 Construction of pMON45631 and pMON45632 Aspergillus Ochraceus NADPH Cytochrome p450 Reductase 0269. A strategy based on two step PCR from an internal BamHI site approximately 170 bp upstream of the 5' splice 0273 Generation of Anti-11-O-hydroxylase Antibodies Site was employed to generate clones lacking the intron. 0274 Polyclonal antibodies against Aspergillus Ochra ceus 11 alpha hydroxylase and NADPH cytochrome p450 Primer A. oxred-for 3: reductase were raised in rabbits against Synthetic peptides GGATCCCTCGCGACCTGTGATCAT (SEQ ID NO: 20) (prepared by Sigma/Genosis, The Woodlands, Tex.) corre Primer A. oxred-for 4: sponding to Several regions of the following predicted CGAAGATTTCTTGTACAAGGATGAATGGAAGACT (SEQ ID NO: 21) protein Sequences: TTTC Primer A. oxred-rev6: 11aOH peptide 1: CTGAAAAGTCTTCCATTCATCCTTGTACAAGAAA (SEQ ID NO: 22) AAAYWLATLQPSDLPELN (SEQ ID NO: 23) TC 11aOH peptide 2: 0270 Primers A.oxred-for4 and rev6 were complemen CRQILTPYIHKRKSLKGTTD (SEQ ID NO: 24) tary and flanked the intron. The first PCR reaction used an 11aoH peptide 3: A.OXred clone linearized at the internal BamHI site as HMGFGHGWHACPGRFFASNEI (SEQ ID NO: 25) template. Polymerase and buffers were supplied by the PCR core kit (Roche Molecular Biochemicals, Indianapolis, oxir peptide 1: Ind.). Primer and dNTP concentrations were 500 nM and CTYWAWAKDPYASAGPAMING (SEQ ID NO: 26) 200 uM, respectively. Two reactions were performed, using US 2005/0003473 A1 Jan. 6, 2005 30
0275] The 11aOH peptide 2 (SEQ ID NO: 24) corre transfection, 800 ul SF-900 medium was added to each tube. sponds to the G helix, G/H loop, and H helix region present The cells were washed one time with 2 ml SF-900 medium, in an alignment of the amino acid Sequence of 11 alpha and the DNA mixtures were added to the cells. The cultures hydroxylase with the corresponding sequence of CYP3A4 were allowed to incubate for 5 hours at 27 C. Following the described by Wang and Lu, (Drug Metab. Dispos. 25(6), 5 hr incubation period, the transfection mixture was 762-767, 1997). The 11aoH peptide 3 (SEQ ID NO: 25) removed and the cultures were replenished with 3 ml per corresponded to the peptide fragment from the heme-bind well IPL-41 medium (Life Technologies) supplemented with ing domain. 10% fetal bovine serum. Following a three day incubation 0276. Immunological grade peptides were monitored for period, the cells were harvested, centrifuged, and the Super purity using reverse phase high performance liquid chroma natant that contained recombinant virus (designated as pas tography (HPLC). Each peptide was conjugated to keyhole Sage 1 or P1 Stock) was removed and stored at 4 C. A larger limpet hemacyanin (KLH) and Suspended in Complete Fre viral stock was made by infecting 100 ml fresh Sf9 cells at und's Adjuvant. The conjugated peptide was then injected 5x10 cells per ml with 0.5 ml of the P1 medium. This larger Subcutaneously at multiple sites into rabbits. Each conju (P2) Stock was then titered using a plaque assay protocol gated peptide was injected into two rabbits. All Subsequent (O'Reilly et al., 1992), and used for production of the 11 immunizations were given in incomplete Freund's Adjuvant. alpha hydroxylase or oxidoreductase enzymes, Separately or In general, five Subsequent injections were given at two in combination with each other. week intervals following the initial immunization. IgG frac 0279 FIG. 14 sets forth an immunoblot illustrating tions were affinity-purified using a Sepharose-Protein A expression of Aspergillus Ochraceus P450 11 alpha hydroxy column. Fractions from the two rabbits injected with each lase in baculovirus-infected insect cells harvested at 25 and peptide were combined at a 1:1 ratio. The pooled anti-11 48 hours post infection. The nitrocellulose membrane was alpha hydroxylase (rabbits GN 1187/1188) was 0.34 mg/ml probed with a 1:1 mixture of antibodies prepared two rabbits IgG. The pooled anti-oxred (rabbits GN 2023/2024) was immunized with a conjugated Synthetic peptide 11aOH 0.26 mg/ml IgG. The combined IgGs were each diluted 1:10, peptide 2 (SEQ ID NO 24). 1:100 and 1:1,000 for a pilot experiment to determine which 0280 FIG. 15 sets forth an immunoblot illustrating was dilution was optimal for probing Western blots. The expression of Aspergillus ochraceus P450 oxidoreductase in 1:10 dilution gave the best results and was used for probing baculovirus-infected insect cells harvested at 25 and 48 Subsequent Westerns. hours post infection. The nitrocellulose membrane was probed with a 1:1 mixture of antibodies prepared two rabbits Example 16 immunized with a conjugated Synthetic peptide OXr peptide Insect Cell Infection and Heterologous Expression 1 (SEQ ID NO 26). 0277 Proteins were expressed in Sf9 insect cells using Example 17 baculovirus shuttle vectors (Luckow et al., J. Virol. 67:4566 4579, 1993). The baculovirus shuttle vector (bacmid) con Co-infection Baculoviruses Expressing of tains a mini-F replicon for expression in bacterial cells, a Aspergillus Ochraceus 11 Alpha Hydroxylase and kanamycin resistance marker for Selection, and attTn7 the Human Oxidoreductase target site for the bacterial TnT transposon) within the lacZO. 0281 Sf9 cells were co-infected with virus particles that Sequence. Each of these elements is inserted into the poly contained the steroid 11 alpha hydroxylase cDNA and a hedrin locus of the Autographa Californica nuclear polyhe separate virus containing a human NADPH P450-oxi drosis virus (AcNPV, the native baculovirus) genome. A doreductase. Both viruses were added at a multiplicity of donor plasmid (pFastBac1, Life Technologies) was used to infection (MOI) ratio of 0.1:0.01 (11 aOH to oxr). One day deliver the gene to be expressed and was inserted into the after infection, 0.9 tug/ml hemin chloride was added to the bacmid via the bacterial Tn7 transposition elements. pFast culture. The cells were harvested by centrifugation three Bac1 contains the TnT. left and right ends flanking the days after infection (unless specified differently), and the polyhedrin promoter, a polylinker cloning Sequence, the washed cell pellets were frozen until processed for Sub SV40 polyA transcription termination Sequence, and the cellular fractions. gentamicin resistance gene for Selection. Recombinant Viruses were generated following transformation of the Example 18 pFastBac1 plasmid, which contained a single 11 alpha hydroxylase or oxidoreductase cDNA, into DH 10Bac E. coli Co-infection Baculoviruses Expressing of cells (Life Technologies) that contained the bacmid and Aspergillus Ochraceus 11 Alpha Hydroxylase and helper plasmid. Aspergillus Ochraceus oxidoreductase 0278 Transfections were performed using CellFectinTM 0282 Sf9 cells are co-infected with virus particles that reagent (Life Technologies) following the manufacturers contain the steroid 11 alpha hydroxylase cDNA and a protocol for Spodoptera frugigperda (Sf9) cells. Cells were separate virus containing A. Ochraceus NADPH P450 seeded in 6-well tissue culture plates at 9x10-cells per well oxidoreductase. Both viruses are added at a multiplicity of in SF-900 serum-free medium (Life Technologies) and infection (MOI) ratio of 0.1:0.01 (11 aOH to oxr). One day allowed to attach for at least one hour. The transfection after infection, 0.9 tug/ml hemin chloride is added to the mixtures were made following the addition of 5ul miniprep culture. The cells are harvested by centrifugation three days DNA and 5 ul Cellfectin to polystyrene tubes that contained after infection (unless specified differently), and the washed 200 ul SF-900 medium. The mixtures were allowed to cell pellets are frozen until needed in Subsequent experi incubate for 15-30 minutes at room temperature. Prior to ments that require processing into for Sub-cellular fractions. US 2005/0003473 A1 Jan. 6, 2005
Example 19 0290 The HPLC system consisted of a Model 1050 Series pump, autoinjector and variable wavelength detector Preparation of Subcellular Fractions from (Hewlett-Packard, Naperville, Ill.), and a Model TC-50 Baculovirus-infected Insect Cells temperature controller and Model CH-30 column heater (both Eppendorf, Madison, Wis.). 0283) One half gram of the cell pastes from infected Sf9 cells and uninfected control cells were thawed and SuS 0291 Cell membrane fractions derived from insect cells pended in 40 ml of 0.25 M Sucrose with 10 mM. KHPO4, transfected with recombinant baculoviruses expressing adjusted to pH 7.4. The Suspensions were homogenized 11-hydroxylase and complementary electron transport pro using a Fisher Sonic Dismembrator, model 300 probe Soni teins were analyzed for 11-hydroxylase activity in a reaction cator (Fisher Scientific, St. Louis, Mo.). The samples were mixture containing 80 mM phosphate buffer, pH 7.4, 8 mM transferred to a conical centrifuge tube (Corning CoStar MgCl, and 0.9 mM NADP in a final volume of 200 ul. In Corporation, Cambridge, Mass.) and Subjected to centrifu order to insure an adequate Source of reducing equivalents, gation at 500xg at 5 C. for 15 minutes. The pellets were an NADPH regenerating system was provided by adding resuspended in the same volume of fresh buffer and viewed glucose-6-phosphate dehydrogenase (1.5 U/ml) and 8 mM under a microscope to confirm complete lysis. Few or no glucose-6-phosphate. Steroid Substrate (e.g., androstenedi whole cells were observed. The Supernatants were then one) was provided at a final concentration of 0.3 mM. subjected to centrifugation at 10,000xg for 30 minutes at 5 Reaction mixtures were incubated at 37 C. for 30 min. The C. to collect mitochondria, Golgi and other Subcellular reactions were terminated by the addition of 200ul methanol organelles. The pellets were resuspended in fresh buffer and and then placed on ice. Samples were pelleted by centrifu subjected to centrifugation at 7,800xg for 30 minutes at 5 gation to remove precipitated protein. C. to collect mitochondria. 0292. On one occasion, the incubation was carried out in 0284. The mitochondrial pellets were resuspended in a volume of 0.5 ml in siliconized polypropylene 1.5 ml buffer as described about and the centrifugation was microcentrifuge tubes at 37 C. for 120 minutes. The repeated. The mitochondrial pellets were resuspended in 2 enzyme, prepared from microSomal or mitochondrial frac ml buffered sucrose solution and stored at -80 C. in 100 ul tions, was added and the Substrate added at a concentration aliquots. of 250 uM (e.g., 25 mM methanol stock solution of AD). The cofactor buffer was 100 mM potassium phosphate, pH 0285) The Supernatants from the original mitochondrial 7.4, 7.5 mM MgCl2, 7.5 mM glucose-6-phosphate, 0.80 mM fractionation were subjected to centrifugation at 200,000xg NADP, and 1.0 units/mL glucose-6-phosphate dehydroge for 1 hour at 5 C. The microSomal pellets were resuspended nase. HPLC samples were prepared by terminating the 0.5 in 2 ml buffered sucrose solution and stored at -80 C. in ml reaction mixture by addition of 0.3 ml methanol, Vor 100 ul aliquots. texing three times for 2 Seconds and Storing on ice. The tubes 0286 Microsomal Incubations were spun for 5 minutes at ~20,000xg in a microcentrifuge and the Samples transferred to autoSampler Vials and capped. 0287 Incubation mixtures consisted of Sf9 microsomes (1.0 mg of protein/mL final concentration), an NADPH 0293 Steroid components present in reaction mixtures generating system and 250 uM Substrate (AD) in 100 mM and media extract were Separated and analyzed by reverse potassium phosphate buffer, pH 7.4 or 150 mM HEPES phase HPLC using a 250 mmx4 mm Vydac analytical C-4 buffer, pH 7.4. The NADPH-generating system was com column. Chromatograms were developed using a Solvent posed of the following at the indicated final concentrations: gradient from 40% to 100% methanol over a ten minute time MgCl2 (7.5 mM), D-glucose-6-phosphate (7.5 mM), NADP period and holding at 100% methanol for 5 minutes before (0.80 mM), and glucose-6-phosphate dehydrogenase (1.0 re-equilibration to initial conditions. The column effluent units/mL). Incubations were carried out for the indicated was monitored for UV absorbance at both 254 and 220 nm. times at 37 C. in a water bath. Following incubation, 0294 Androstenedione, testosterone and monohydroxy reactions were terminated by the addition of 0.3 ml metha lated androstenedione metabolites were resolved on a Nova nol. The Samples were Vortexed three times for two Seconds pak C18 column, 4 micron, 3.9x150 mm (Waters Chroma and placed on ice, or Stored at -70 C. for later analysis. tography, Milford, Mass.) equipped with a 0.22 micron Rheodyne precolumn filter at 40 C. and 1.0 ml mobile Example 20 phase/min. A Stepped gradient was utilized with water as mobile phase solvent A and methanol as solvent B. The HPLC Assays to Measure Conversion of Steroid initial concentration of Solvent B was 42% for 6 min. The Substrates to Their Hydroxylated Counterparts percentage of B was increased linearly to 45% over 4 Liquid Chromatography minutes and then held for 3 minutes. The percentage of B 0288 High Performance was then increased linearly to 80% over 10 minutes and held (HPLC) there for an additional 2 minutes for a total run time of 25 0289. The HPLC method used to separate hydroxylated minutes. The ultraviolet detection wavelength was 247 nm Steroid compounds from Steroid Substrates, Such as 11C.- and the injection volume was 200 ul. hydroxyandrostenedione from androstenedione, is a modi 0295) Both the “mitochondria” sample and the “microso fied version of the testosterone hydroxylase assay, described mal' Sample produced peaks matching the HPLC retention by Sonderfan et al., Arch. Biochem. BiophyS. 255:27-41, time of the 11C.-hydroxyandrostenedione Standard, while 1987). The standards for androstenedione and 11-beta-hy other fractions did not. These “mitochondria' and “microso droxyandrostenedione were obtained from Sigma. 11-alpha mal” peaks were 3.2 and 2.3%, respectively, of the total peak hydroxyandrostenedione (89.5% pure, with the major impu area quantitated at 247 nm. The 11C.-hydroxyandrostenedi rity being androstenedione) was provided by Searle one Standard was also Spiked into a blank microSomal Medicinal Chemistry. HPLC grade water and methanol were incubation Sample at a concentration of 5.0 lig/mL. The obtained from Burdick & Jackson. concentration of the “mitochondria' and “microsomal’ 11C.- US 2005/0003473 A1 Jan. 6, 2005 32 hydroxyandrostenedione peaks were 1.75 and 1.31 lug/mL, 0299 Briefly, genomic DNA is prepared and shotgun after correcting for the purity of the standard (89.5%). These cloned into low copy artificial chromosomes propagated in concentrations represent 2.3 and 1.7% of substrate converted bacteria. A large number of clones are Sequenced to ensure to 11O-hydroxyandrostenedione, using a Substrate concen Statistical representation of the entire genome, and the tration of 250 uM. Sequences of Overlapping clones merged to produce the final map and Sequence of the genome. Analysis of the open 0296 FIG. 16 sets forth an HPLC tracing illustrating the reading frames, will reveal regions which are homologous to conversion of androstenedione (AD) to its 11 alpha hydroxy the Steroid hydroxylase and oxidoreductase genes of the counterpart after incubating AD with Subcellular fractions present invention, and regions of the translated open reading prepared from baculovirus-infected insect cells expressing frames which are homologous to these enzymes using Aspergillus OchraceuS 11 alpha hydroxylase and human programs designed to facilitate multiple Sequence align oxidoreductase. ments of nucleotide and protein Sequence data Such as BLAST, CLUSTALW, and Box.Shade. Genes which encode Example 21 these proteins are obtained from the artificial chromosomes and recloned into expression vectorS Such as pFastBac1, Recognition of Aspergillus OchraceuS 11 Alpha transformed into appropriate host cells, which are assayed Hydroxylase and Aspergillus ochraceus NADPH for the presence of enzymes capable of carrying out the Cytochrome p450 Reductase by Immunoblotting conversion of Steroid Substrates to their oxidized counter Using Polyclonal Antibodies Generated Against parts. Synthetic Peptides 0300. It is intended that the scope of the present invention 0297 Proteins from Sf9 cell lysates (obtained from unin be determined by reference to the appended claims. It is fected and recombinant baculovirus-infected cells) were recognized that a number of variations can be made to this loaded onto lanes of a 10% gradient acrylamide mini gel invention as it is currently described but which do not depart (Biorad, Hercules, Calif.) at equal concentrations (10 ug per from the Scope and Spirit of the invention without compro well). The proteins were separated by electrophoresis at 16 mising any of its advantages. These include isolation of mAmps constant current for approximatley 1 hr in a Tris homologous genes from microorganisms known to carry out glycine buffer containing 0.1% SDS (Sigma, St. Louis, 11 alpha hydroxylation of Steroid Substrates, preferably fungi and bacteria. This invention is also directed to any Mo.). The proteins were transferred to nitrocellulose (Schle Substitution of analogous components. This includes, but is icher & Schuell, Keene, N.H.) for 40 min at 70 mAmp not restricted to use of these techniques to isolate other constant current. Primary antibodies were diluted 1:10 (from P450s which are involved in steroidogenesis, including Stock concentrations of 0.34 mg/ml IgG for anti-11 alpha hydroxylases that act at other positions in the core molecule, hydroxylase (antibodies GN-1187 and GN-1188 prepared and use of these enzymes to facilitate bioconversion of from peptide 11a(OH peptide 2 CRQILTPYIHKRK Steroid intermediates in modified host microorganisms. SLKGTTD (SEQ ID NO: 24)), and 0.26 mg/ml IgG for anti-oxred (antibodies GN-2023 and GN-12024 prepared 0301 All references, patents, or applications cited herein from oxr peptide 1 CTYWAVAKDPYASAGPAMNG (SEQ are incorporated by reference in their entirety, as if written ID NO: 26)) and used to probe the nitrocellulose membrane. herein. The antigens were detected using anti-rabbit horseradish peroxidase (HRP)-linked secondary antibody as recom References mended by the manufacturer (New England Biolabs, Bev erly, Mass.). Chemilumiescence was detected using luminol 0302) Altschul S F; Gish W; Miller W: Myers E W: and peroxide reagents (New England Biolabs, Beverly, Lipman DJ Basic local alignment Search tool. J. Molec. Mass.) following the protocol provided by the vendor. Light Biol. (Oct. 5, 1990), 215(3), 403-10. emission was recorded using X-OMAT AR film (Eastman 0303 Anfossi et al., Proc. Natl. Acad. Sci. (U.S.A.) Kodak Company, Rochester, N.Y.). Images were recorded 86:3379-3383 (1989) using a Minolta Dimage V digital camera (Minolta Corpo 0304) Arfin et al. Proc. Natl. Acad. Sci. (U.S.A.) 92:7714 ration, Ramsey, N.J.). 7718 (1995) Example 22 0305 Armour, et al., FEBS Lett. 307:113-115 (1992) Characterization of the Aspergillus OchraceuS 0306 Baldwin et al., Gene Ther. 4:1142-1149 (1997) Genomic DNA Encoding 11 Alpha Hydroxylase 0307 Barany, Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193 and Oxidoreductase (1991) 0298 The approaches described above can be used to 0308 Bassat et al., J. Bacteriol. 169:751-757 (1987) facilitate the identification of genes encoding Steroid hydroxylases and oxidoreductases within the genome of 0309 Baum et al., J. Hematother. 5: 323-329 (1996) Aspergillus OchraceuS and closely related microorganisms, 0310. Becker et al., EMBO J. 8:3685-3691 (1989) including Aspergillus niger and Aspergillus nidulans. Other preferred organisms are Rhizopus Oryzae, Rhizopus Stoloni 0311 Ben-Bassat et al., J. Bacteriol. 169:751–757 (1987) fer, Streptomyces fradiae, Bacillus megaterium, Pseudomo 0312 Berkner, BioTechniques 6:616-629 (1988) naS cruciviae, Trichothecium roseum, Fusarium Oxysporum f Sp. Cepae, Rhizopus arrhizus, and Monosporium Oliva 0313 Berkner, Current Top. Microbiol. Immunol. ceum. Other preferred organisms that are known to have 158:39-66 (1992) Steroid 11 alpha hydroxylase activity are described in the 0314 Blobel and Dobberstein, J. Cell Biol. 67:835-851 detailed description of the invention, above. (1975) US 2005/0003473 A1 Jan. 6, 2005 33
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SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 68 <210> SEQ ID NO 1 &2 11s LENGTH 1774 &212> TYPE DNA <213> ORGANISM: Aspergillus ochraceus &22O > FEATURE <221 NAME/KEY: CDS <222> LOCATION: (146) . . (1690) <223> OTHER INFORMATION: Aspergillus ochraceus 11 alpha hydroxylase <400 SEQUENCE: 1 tggaagttitt tacacttatt at gcc.ggagc cqaaagattic toagtc gagg ggttggggaa 60 caa.cactata agacctacaa coactitggat ttggtgaatt tacacgggca ttatcaaaac 120 agccacaagc togacagotca ttatc atg ccc titc titc act ggg citt citg gcg 172 Met Pro Phe Phe Thr Gly Leu Leu Ala 1 5
att tac cat agt citc ata citc gac aac cca gtc. caa acc citg agc acc 220 Ile Tyr His Ser Leu Ile Leu Asp Asn Pro Val Glin Thr Leu Ser Thr 10 15 2O 25
att gtc gta ttg gog go a gog tac togg citc goa acg citc Cag CC g agc 268 Ile Val Val Lieu Ala Ala Ala Tyr Trp Leu Ala Thr Lieu Glin Pro Ser 30 35 40
gac citt cott gag citg aat coc goc aaa cca titc gag titc acc aat cqt 316 Asp Leu Pro Glu Lieu. Asn Pro Ala Lys Pro Phe Glu Phe Thr Asn Arg 45 5 O 55
US 2005/0003473 A1 Jan. 6, 2005 38
-continued cag cqt citc agg cct acg citt citc ggc toc titt cqt cqg cag goa acg 276 Glin Arg Lieu Arg Pro Thr Lieu Lieu Gly Ser Phe Arg Arg Glin Ala Thr 365 370 375 aat gac atc aag citg aag agc ggg titt gtc. ata aag aaa ggg act aga 324 Asn Asp Ile Lys Lieu Lys Ser Gly Phe Val Ile Lys Lys Gly. Thir Arg 38O 385 390 gto gtg atc gac agc acc cat at g tog aat coc gag tat tac act gac 372 Val Val Ile Asp Ser Thr His Met Trp Asin Pro Glu Tyr Tyr Thr Asp 395 400 405 cct citc cag tac gac ggg tac cqc tac titc aac aag cqg cag aca coc 420 Pro Leu Gln Tyr Asp Gly Tyr Arg Tyr Phe Asn Lys Arg Glin Thr Pro 410 415 420 425 ggc gag gac aag aac gog titg citc gtc agc aca agc gcc aac cac atg 468 Gly Glu Asp Lys Asn Ala Leu Lieu Val Ser Thr Ser Ala Asn His Met 430 435 4 40 gga titc ggt cac ggc gtt cac goc tot cott ggc aga titc ttic goc to c 516 Gly Phe Gly His Gly Val His Ala Cys Pro Gly Arg Phe Phe Ala Ser 445 450 455 aac gag atc aag att gcc titg tot cat atc atc tta aat tat gag togg 564 Asn Glu Ile Lys Ile Ala Lieu. Cys His Ile Ile Lieu. Asn Tyr Glu Trip 460 465 470 cgt citt coa gac ggc titc aag coc cag cott citc aac atc ggg at g act 612 Arg Lieu Pro Asp Gly Phe Lys Pro Glin Pro Leu Asn. Ile Gly Met Thr 475 480 485 tat citg gog gat coc aat acc agg at g citg atc agg cca cqc aag gog 660 Tyr Lieu Ala Asp Pro Asn. Thir Arg Met Lieu. Ile Arg Pro Arg Lys Ala 490 495 5 OO 505 tat citg gog gat coc aat acc agg at g citg atcaggccac goaaggcgga 710 Tyr Lieu Ala Asp Pro Asn. Thir Arg Met Lieu 510 515 agtgttattg gtcagtgggit galagcaagtc gcagaaatgt gtaacaattt ataagaataa 770 aaaa. 774
<210> SEQ ID NO 2 &2 11s LENGTH 515 &212> TYPE PRT <213> ORGANISM: Aspergillus ochraceus <400 SEQUENCE: 2 Met Pro Phe Phe Thr Gly Leu Leu Ala Ile Tyr His Ser Leu Ile Leu 1 5 10 15 Asp Asin Pro Val Glin Thr Leu Ser Thr Ile Val Val Leu Ala Ala Ala 2O 25 30 Tyr Trp Lieu Ala Thr Lieu Gln Pro Ser Asp Leu Pro Glu Lieu. Asn Pro 35 40 45 Ala Lys Pro Phe Glu Phe Thr Asn Arg Arg Arg Val His Glu Phe Val 50 55 60 Glu Asn. Ser Lys Ser Lieu Lleu Ala Arg Gly Arg Glu Lieu. His Gly. His 65 70 75 8O Glu Pro Tyr Arg Leu Met Ser Glu Trp Gly Ser Leu Ile Val Leu Pro 85 90 95 Pro Glu Cys Ala Asp Glu Lieu Arg Asn Asp Pro Arg Met Asp Phe Glu 100 105 110 Thr Pro Thr Thr Asp Asp Ser His Gly Tyr Ile Pro Gly Phe Asp Ala 115 120 125 US 2005/0003473 A1 Jan. 6, 2005 39
-continued
Lieu. Asn Ala Asp Pro Asn Lieu. Thir Lys Val Val Thr Lys Tyr Lieu. Thr 130 135 1 4 0 Lys Ala Lieu. Asn Lys Lieu. Thir Ala Pro Ile Ser His Glu Ala Ser Ile 145 15 O 155 160 Ala Met Lys Ala Val Lieu Gly Asp Asp Pro Asp Trp Arg Glu Ile Tyr 1.65 170 175 Pro Ala Arg Asp Leu Lieu Gln Leu Val Ala Arg Met Ser Thr Arg Val 18O 185 19 O Phe Leu Gly Glu Glu Met Cys Asn. Asn Glin Asp Trp Ile Glin Thr Ser 195 200 2O5 Ser Glin Tyr Ala Ala Leu Ala Phe Gly Val Gly Asp Lys Lieu Arg Ile 210 215 220 Tyr Pro Arg Met Ile Arg Pro Ile Val His Trp Phe Met Pro Ser Cys 225 230 235 240 Trp Glu Lieu Arg Arg Ser Leu Arg Arg Cys Arg Glin Ile Lieu. Thr Pro 245 250 255 Tyr Ile His Lys Arg Lys Ser Lieu Lys Gly Thr Thr Asp Glu Glin Gly 260 265 27 O Lys Pro Leu Met Phe Asp Asp Ser Ile Glu Trp Phe Glu Arg Glu Lieu 275 280 285 Gly Pro Asn His Asp Ala Wall Leu Lys Glin Val Thr Lieu Ser Ile Val 29 O 295 3OO Ala Ile His Thir Thr Ser Asp Leu Lleu Lieu Glin Ala Met Ser Asp Lieu 305 310 315 320 Ala Glin Asn Pro Lys Wall Leu Glin Ala Val Arg Glu Glu Val Val Arg 325 330 335 Val Lieu Ser Thr Glu Gly Lieu Ser Lys Val Ser Lieu. His Ser Lieu Lys 340 345 35 O Leu Met Asp Ser Ala Lieu Lys Glu Ser Glin Arg Lieu Arg Pro Thr Lieu 355 360 365 Leu Gly Ser Phe Arg Arg Glin Ala Thr Asn Asp Ile Lys Lieu Lys Ser 370 375 38O Gly Phe Val Ile Lys Lys Gly Thr Arg Val Val Ile Asp Ser Thr His 385 390 395 400 Met Trp Asin Pro Glu Tyr Tyr Thr Asp Pro Leu Gln Tyr Asp Gly Tyr 405 410 415 Arg Tyr Phe Asn Lys Arg Glin Thr Pro Gly Glu Asp Lys Asn Ala Lieu 420 425 43 O Leu Val Ser Thr Ser Ala Asn His Met Gly Phe Gly His Gly Val His 435 4 40 4 45 Ala Cys Pro Gly Arg Phe Phe Ala Ser Asn. Glu Ile Lys Ile Ala Lieu 450 455 460 Cys His Ile Ile Leu Asn Tyr Glu Trp Arg Lieu Pro Asp Gly Phe Lys 465 470 475 480 Pro Glin Pro Leu Asn Ile Gly Met Thr Tyr Leu Ala Asp Pro Asn Thr 485 490 495 Arg Met Lieu. Ile Arg Pro Arg Lys Ala Tyr Lieu Ala Asp Pro Asn Thr 5 OO 505 51O.
Arg Met Lieu 515 US 2005/0003473 A1 Jan. 6, 2005 40
-continued
<210> SEQ ID NO 3 &2 11s LENGTH 514 &212> TYPE PRT <213> ORGANISM: Aspergillus ochraceus <400 SEQUENCE: 3 Met Pro Phe Phe Thr Gly Leu Leu Ala Ile Tyr His Ser Leu Ile Leu 1 5 10 15 Asp Asin Pro Val Glin Thr Leu Ser Thr Ile Val Val Leu Ala Ala Ala 2O 25 30 Tyr Trp Lieu Ala Thr Lieu Gln Pro Ser Asp Leu Pro Glu Lieu. Asn Pro 35 40 45 Ala Lys Pro Phe Glu Phe Thr Asn Arg Arg Arg Val His Glu Phe Val 50 55 60 Glu Asn. Ser Lys Ser Lieu Lleu Ala Arg Gly Arg Glu Lieu. His Gly. His 65 70 75 8O Glu Pro Tyr Arg Leu Met Ser Glu Trp Gly Ser Leu Ile Val Leu Pro 85 90 95 Pro Glu Cys Ala Asp Glu Lieu Arg Asn Asp Pro Arg Met Asp Phe Glu 100 105 110 Thr Pro Thr Thr Asp Asp Ser His Gly Tyr Ile Pro Gly Phe Asp Ala 115 120 125 Leu Asn Ala Asp Pro Asn Lieu. Thr Lys Val Val Thr Lys Tyr Leu Thr 130 135 1 4 0 Lys Ala Lieu. Asn Lys Lieu. Thir Ala Pro Ile Ser His Glu Ala Ser Ile 145 15 O 155 160 Ala Met Lys Ala Val Lieu Gly Asp Asp Pro Asp Trp Arg Glu Ile Tyr 1.65 170 175 Pro Ala Arg Asp Leu Lieu Gln Leu Val Ala Arg Met Ser Thr Arg Val 18O 185 19 O Phe Leu Gly Glu Glu Met Cys Asn. Asn Glin Asp Trp Ile Glin Thr Ser 195 200 2O5 Ser Glin Tyr Ala Ala Leu Ala Phe Gly Val Gly Asp Lys Lieu Arg Ile 210 215 220 Tyr Pro Arg Met Ile Arg Pro Ile Val His Trp Phe Met Pro Ser Cys 225 230 235 240 Trp Glu Lieu Arg Arg Ser Leu Arg Arg Cys Arg Glin Ile Lieu. Thr Pro 245 250 255 Tyr Ile His Lys Arg Lys Ser Lieu Lys Gly Thr Thr Asp Glu Glin Gly 260 265 27 O Lys Pro Leu Met Phe Asp Asp Ser Ile Glu Trp Phe Glu Arg Glu Lieu 275 280 285 Gly Pro Asn His Asp Ala Wall Leu Lys Glin Val Thr Lieu Ser Ile Val 29 O 295 3OO Ala Ile His Thir Thr Ser Asp Leu Lleu Lieu Glin Ala Met Ser Asp Lieu 305 310 315 320 Ala Glin Asn Pro Lys Wall Leu Glin Ala Val Arg Glu Glu Val Val Arg 325 330 335 Val Lieu Ser Thr Glu Gly Lieu Ser Lys Val Ser Lieu. His Ser Lieu Lys 340 345 35 O Leu Met Asp Ser Ala Lieu Lys Glu Ser Glin Arg Lieu Arg Pro Thr Lieu 355 360 365 US 2005/0003473 A1 Jan. 6, 2005 41
-continued
Leu Gly Ser Phe Arg Arg Glin Ala Thr Asn Asp Ile Lys Lieu Lys Ser 370 375 38O Gly Phe Val Ile Lys Lys Gly Thr Arg Val Val Ile Asp Ser Thr His 385 390 395 400 Met Trp Asin Pro Glu Tyr Tyr Thr Asp Pro Leu Gln Tyr Asp Gly Tyr 405 410 415 Arg Tyr Phe Asn Lys Arg Glin Thr Pro Gly Glu Asp Lys Asn Ala Lieu 420 425 43 O Leu Val Ser Thr Ser Ala Asn His Met Gly Phe Gly His Gly Val His 435 4 40 4 45 Ala Cys Pro Gly Arg Phe Phe Ala Ser Asn. Glu Ile Lys Ile Ala Lieu 450 455 460 Cys His Ile Ile Leu Asn Tyr Glu Trp Arg Lieu Pro Asp Gly Phe Lys 465 470 475 480 Pro Glin Pro Leu Asn Ile Gly Met Thr Tyr Leu Ala Asp Pro Asn Thr 485 490 495 Arg Met Lieu. Ile Arg Pro Arg Lys Ala Glu Ile Asp Met Ala Ser Lieu 5 OO 505 51O.
Thir Wall
<210> SEQ ID NO 4 &2 11s LENGTH 2.031 &212> TYPE DNA <213> ORGANISM: homo sapiens &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (1) . . (2031) <223> OTHER INFORMATION: human oxidoreductase
<400 SEQUENCE: 4 atg gga gac toc cac gtg gac acc agc toc acc gtg toc gag gog gtg 48 Met Gly Asp Ser His Val Asp Thr Ser Ser Thr Val Ser Glu Ala Val 1 5 10 15 gcc gala gaa gta tot citt titc agc at g acg gac atg att citg ttt tog 96 Ala Glu Glu Val Ser Leu Phe Ser Met Thr Asp Met Ile Leu Phe Ser 2O 25 30 citc atc gtg ggit citc cita acc tac togg titc citc titc aga aag aaa aaa 144 Lieu. Ile Val Gly Lieu Lieu. Thir Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45 gala gaa gtc. ccc gag titc acc aaa att cag aca ttg acc toc tot gtc 192 Glu Glu Val Pro Glu Phe Thr Lys Ile Glin Thr Leu Thir Ser Ser Val 50 55 60 aga gag agc agc titt gtg gala aag atg aag aaa acg ggg agg aac atc 240 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn. Ile 65 70 75 8O atc gtg titc tac ggc ticc cag acg ggg act gca gag gag titt gcc aac 288 Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 cgc citg to c aag gac goc cac cqc tac ggg at g c ga ggc at g to a gog 336 Arg Lieu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 gac cott gag gag tat gac citg goc gac citg agc agc ctd coa gag atc 384 Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 gac aac goc ct g g to gtt ttctgc at g goc acc tac ggit gag gga gac 432 Asp Asn Ala Lieu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp
US 2005/0003473 A1 Jan. 6, 2005 44
-continued Lieu. Ile Val Gly Lieu Lieu. Thir Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45 Glu Glu Val Pro Glu Phe Thr Lys Ile Glin Thr Leu Thir Ser Ser Val 50 55 60 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn. Ile 65 70 75 8O Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Lieu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Asn Ala Lieu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 1 4 0 Pro Thr Asp Asn Ala Glin Asp Phe Tyr Asp Trp Leu Glin Glu Thir Asp 145 15 O 155 160 Val Asp Leu Ser Gly Wall Lys Phe Ala Val Phe Gly Lieu Gly Asn Lys 1.65 170 175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Lieu 18O 185 19 O Glu Gln Leu Gly Ala Glin Arg Ile Phe Glu Lieu Gly Lieu Gly Asp Asp 195 200 2O5 Asp Gly Asn Lieu Glu Glu Asp Phe Ile Thir Trp Arg Glu Glin Phe Trp 210 215 220 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240 Ser Ile Arg Glin Tyr Glu Lieu Val Val His Thr Asp Ile Asp Ala Ala 245 250 255 Lys Val Tyr Met Gly Glu Met Gly Arg Lieu Lys Ser Tyr Glu Asn Glin 260 265 27 O Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Lieu. Asn Glin Gly Thr Glu Arg His Leu Met His Leu Glu 29 O 295 3OO Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Ala Lieu Val Asn Gln Leu Gly Lys 325 330 335 Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Lieu. Asn. Asn Lieu. Asp 340 345 35 O Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 38O Val Leu Tyr Glu Leu Ala Glin Tyr Ala Ser Glu Pro Ser Glu Glin Glu 385 390 395 400 Leu Lieu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Lieu. Tyr 405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Glin 420 425 43 O Asp Cys Pro Ser Leu Arg Pro Pro Ile Asp His Lieu. Cys Glu Lieu Lieu US 2005/0003473 A1 Jan. 6, 2005 45
-continued
435 4 40 4 45 Pro Arg Lieu Glin Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Wal 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465 470 475 480 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr Asn Trp Lieu Arg Ala 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu Val Pro Met Phe 5 OO 505 51O. Val Arg Lys Ser Glin Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro Val 515 52O 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly Phe Ile 530 535 540 Glin Glu Arg Ala Trp Lieu Arg Glin Glin Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Lleu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr Lieu. Tyr Arg 565 570 575 Glu Glu Lieu Ala Glin Phe His Arg Asp Gly Ala Lieu. Thr Glin Lieu. Asn 58O 585 59 O Val Ala Phe Ser Arg Glu Gln Ser His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys Glin Asp Arg Glu His Leu Trp Llys Leu. Ile Glu Gly Gly Ala 610 615 62O His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Val Glin 625 630 635 640 Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly Ala Met Glu His Ala 645 650 655 Glin Ala Val Asp Tyr Ile Lys Lys Lieu Met Thr Lys Gly Arg Tyr Ser 660 665 67 O Leu Asp Val Trp Ser 675
<210> SEQ ID NO 6 &2 11s LENGTH 677 &212> TYPE PRT <213> ORGANISM: homo sapiens <400 SEQUENCE: 6 Met Gly Asp Ser His Val Asp Thr Ser Ser Thr Val Ser Glu Ala Val 1 5 10 15 Ala Glu Glu Val Ser Leu Phe Ser Met Thr Asp Met Ile Leu Phe Ser 2O 25 30 Lieu. Ile Val Gly Lieu Lieu. Thir Tyr Trp Phe Leu Phe Arg Lys Lys Lys 35 40 45 Glu Glu Val Pro Glu Phe Thr Lys Ile Glin Thr Leu Thir Ser Ser Val 50 55 60 Arg Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn. Ile 65 70 75 8O Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Lieu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 US 2005/0003473 A1 Jan. 6, 2005 46
-continued Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Asn Ala Lieu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 1 4 0 Pro Thr Asp Asn Ala Glin Asp Phe Tyr Asp Trp Leu Glin Glu Thir Asp 145 15 O 155 160 Val Asp Leu Ser Gly Wall Lys Phe Ala Val Phe Gly Lieu Gly Asn Lys 1.65 170 175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Lys Arg Lieu 18O 185 19 O Glu Gln Leu Gly Ala Glin Arg Ile Phe Glu Lieu Gly Lieu Gly Asp Asp 195 200 2O5 Asp Gly Asn Lieu Glu Glu Asp Phe Ile Thir Trp Arg Glu Glin Phe Trp 210 215 220 Pro Ala Val Cys Glu His Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240 Ser Ile Arg Glin Tyr Glu Lieu Val Val His Thr Asp Ile Asp Ala Ala 245 250 255 Lys Val Tyr Met Gly Glu Met Gly Arg Lieu Lys Ser Tyr Glu Asn Glin 260 265 27 O Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Lieu. Asn Glin Gly Thr Glu Arg His Leu Met His Leu Glu 29 O 295 3OO Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Ala Lieu Val Asn Gln Leu Gly Lys 325 330 335 Ile Leu Gly Ala Asp Leu Asp Val Val Met Ser Lieu. Asn. Asn Lieu. Asp 340 345 35 O Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Ser Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 38O Val Leu Tyr Glu Leu Ala Glin Tyr Ala Ser Glu Pro Ser Glu Glin Glu 385 390 395 400 Leu Lieu Arg Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Lieu. Tyr 405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Glin 420 425 43 O Asp Cys Pro Ser Leu Arg Pro Pro Ile Asp His Lieu. Cys Glu Lieu Lieu 435 4 40 4 45 Pro Arg Lieu Glin Ala Arg Tyr Tyr Ser Ile Ala Ser Ser Ser Lys Wal 450 455 460 His Pro Asn Ser Val His Ile Cys Ala Val Val Val Glu Tyr Glu Thr 465 470 475 480 Lys Ala Gly Arg Ile Asn Lys Gly Val Ala Thr Asn Trp Lieu Arg Ala 485 490 495 Lys Glu Pro Ala Gly Glu Asn Gly Gly Arg Ala Leu Val Pro Met Phe 5 OO 505 51O. Val Arg Lys Ser Glin Phe Arg Leu Pro Phe Lys Ala Thr Thr Pro Val US 2005/0003473 A1 Jan. 6, 2005 47
-continued
515 52O 525 Ile Met Val Gly Pro Gly Thr Gly Val Ala Pro Phe Ile Gly Phe Ile 530 535 540 Glin Glu Arg Ala Trp Lieu Arg Glin Glin Gly Lys Glu Val Gly Glu Thr 545 550 555 560 Leu Lleu Tyr Tyr Gly Cys Arg Arg Ser Asp Glu Asp Tyr Lieu. Tyr Arg 565 570 575 Glu Glu Lieu Ala Glin Phe His Arg Asp Gly Ala Lieu. Thr Glin Lieu. Asn 58O 585 59 O Val Ala Phe Ser Arg Glu Gln Ser His Lys Val Tyr Val Gln His Leu 595 600 605 Leu Lys Glin Asp Arg Glu His Leu Trp Llys Lieu. Ile Glu Gly Gly Ala 610 615 62O His Ile Tyr Val Cys Gly Asp Ala Arg Asn Met Ala Arg Asp Val Glin 625 630 635 640 Asn Thr Phe Tyr Asp Ile Val Ala Glu Leu Gly Ala Met Glu His Ala 645 650 655 Glin Ala Val Asp Tyr Ile Lys Lys Lieu Met Thr Lys Gly Arg Tyr Ser 660 665 67 O Leu Asp Val Trp Ser 675
<210 SEQ ID NO 7 <211& LENGTH 2.322 &212> TYPE DNA <213> ORGANISM: Aspergillus ochraceus &220s FEATURE <221 NAME/KEY: CDS <222> LOCATION: (233) . . (2320) <223> OTHER INFORMATION: Aspergillus ochraceus oxidoreductase <400 SEQUENCE: 7 cittattitcgt ttaggaagag caccggctitc ggtgtc.ctitc cittacccitct tattottcct 60 cittctgactic cotttttgtt attgatc.gcc catctoggtg aacatttggg atatotttcc 120 citctoccoct cocgcc.ccga cccitcctitat cittctoctoc cqtccagoat ttagotc.gc.c 18O atcgaatticg caattic ctitc citcgtgactic titcatc.gctd agcgtc.citca to at g g c g 238 Met Ala 1 caa citc gat act citc gat ttg gttc gtc ct g g to go.g. citc ttg gtg ggit 286 Glin Lieu. Asp Thr Lieu. Asp Leu Val Val Lieu Val Ala Lieu Lieu Val Gly 5 10 15 agc gtg gC c tac titc acc aag ggc acc tac togg gcc gtc goc aaa gac 334 Ser Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp Ala Val Ala Lys Asp 2O 25 30 cct tat gcc to g g ct ggit cog gog atgaat gga ggc gcc aag goc ggc 382 Pro Tyr Ala Ser Ala Gly Pro Ala Met Asn Gly Gly Ala Lys Ala Gly 35 40 45 50 aag act cqc gac att gtt cag aaa at g gac gala act ggc aaa aac tot 430 Lys Thr Arg Asp Ile Val Glin Lys Met Asp Glu Thr Gly Lys Asn. Cys 55 60 65 gtg att titc tac ggc ticg caa acc ggt acc got gag gac tac gog to c 478 Val Ile Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Asp Tyr Ala Ser 70 75 8O aga citg gC c aag gaa goc tocc cag cqa titc ggit citc aag acc at g g to 526 Arg Lieu Ala Lys Glu Gly Ser Glin Arg Phe Gly Lieu Lys Thr Met Val
US 2005/0003473 A1 Jan. 6, 2005 50
-continued
695
SEQ ID NO 8 LENGTH 695 TYPE ORGANISM: Aspergillus ochraceus
<400 SEQUENCE: 8
Met Ala Glin Leu Asp Thr Teu Asp Telu Wall Wall Teu Wall Ala Telu Telu 1 5 10 15
Wall Gly Ser Wall Ala Phe Thr Lys Gly Thr Trp Ala Wall Ala 25 30
Asp Pro Ala Ser Ala Gly Pro Ala Met Asn Gly Gly Ala 35 40 45
Ala Gly Lys Thr Asp Ile Wall Glin Met Asp Glu Thr Gly 50 55 60
Asn Cys Wall Ile Phe Tyr Gly Ser Glin Thr Gly Thr Ala Glu Asp Tyr 65 70 75
Ala Ser Arg Telu Ala Glu Gly Ser Glin Arg Phe Gly Telu Lys Thr 85 90 95
Met Wall Ala Asp Teu Glu Tyr Asp Glu Asn Teu Glu Phe 100 105 110
Pro Glu Asp Wall Wall Phe Phe Wall Telu Ala Thr Tyr Gly Glu Gly 115 120 125
Glu Pro Thr Asp Asn Ala Wall Glu Phe Glin Phe Wall Thr Gly Glu 130 135 1 4 0
Asp Ala Ala Phe Glu Ser Gly Ala Thr Ala Asp Asp Pro Telu Ser 145 15 O 155 160
Ser Telu Wall Thr Phe Gly Telu Gly Asn Asn Thr Glu His 1.65 170 175
Asn Ala Met Wall Arg Asn Wall Asp Ala Ala Teu Thr Lys Phe Gly 18O 185 19 O
Ala Glin Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr 195 200
Met Glu Glu Asp Phe Teu Ala Trp Lys Glu Pro Met Trp Ala Ala Telu 210 215 220
Ser Glu Ala Met Asn Teu Glin Glu Arg Asp Ala Wall Glu Pro Wall 225 230 235 240
Phe Asn Wall Thr Glu Asp Glu Ser Telu Ser Pro Glu Asp Glu Asn Wall 245 250 255
Telu Gly Glu Pro Thr Glin Gly His Telu Glin Gly Glu Pro Gly 260 265 27 O
Pro Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ser Glu Ser Arg 275 280 285
Glu Telu Phe Asn Wall Asp Arg Asn Teu His Met Glu Ile Ser 29 O 295
Ile Ala Gly Ser Asn Teu Thr Tyr Glin Thr Gly Asp His Ile Ala Wall 305 310 315 320
Trp Pro Thr Asn Ala Gly Ser Glu Wall Asp Arg Phe Teu Glin Ala Phe 325 330 335
Gly Telu Glu Gly Lys His Ser Wall Ile Asn Ile Gly Ile Asp 340 345 35 O US 2005/0003473 A1 Jan. 6, 2005 51
-continued Val Thr Ala Lys Val Pro Ile Pro Thr Pro Thir Thr Tyr Asp Ala Ala 355 360 365 Val Arg Tyr Tyr Leu Glu Val Cys Ala Pro Val Ser Arg Glin Phe Val 370 375 38O Ser Thr Lieu Ala Ala Phe Ala Pro Asp Glu Ala Thr Lys Ala Glu Ile 385 390 395 400 Val Arg Lieu Gly Gly Asp Lys Asp Tyr Phe His Glu Lys Ile Thr Asn 405 410 415 Arg Cys Phe Asn. Ile Ala Glin Ala Lieu Glin Ser Ile Thr Ser Lys Pro 420 425 43 O Phe Thr Ala Val Pro Phe Ser Leu Leu Ile Glu Gly Ile Thr Lys Leu 435 4 40 4 45 Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Glin Lys Asp 450 455 460 Lys Ile Ser Ile Thr Ala Val Val Glu Ser Val Arg Leu Pro Gly Glu 465 470 475 480 Glu His Ile Val Lys Gly Val Thir Thr Asn Tyr Lieu Lleu Ala Lieu Lys 485 490 495 Glu Lys Glin Asn Gly Glu Pro Ser Pro Asp Pro His Gly Leu Thr Tyr 5 OO 505 51O. Ser Ile Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Val Pro Val 515 52O 525 His Val Arg His Ser Asn. Phe Lys Lieu Pro Ser Asp Pro Ser Arg Pro 530 535 540 Val Ile Met Val Gly Pro Gly. Thr Gly Val Ala Pro Phe Arg Gly Phe 545 550 555 560 Ile Glin Glu Arg Ala Ala Lieu Ala Ala Lys Gly Glu Lys Val Gly Thr 565 570 575 Thr Lieu Lieu Phe Phe Gly Cys Arg Lys Ser Asp Glu Asp Phe Leu Tyr 58O 585 59 O Lys Asp Glu Trip Lys Thr Phe Glin Glu Gln Leu Gly Asp Ser Lieu Lys 595 600 605 Ile Ile Thr Ala Phe Ser Arg Glu Ser Ala Glu Lys Val Tyr Val Glin 610 615 62O His Arg Lieu Arg Glu His Ala Glu Lieu Val Ser Asp Lieu Lleu Lys Glin 625 630 635 640 Lys Ala Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu 645 650 655 Val Asn Lieu Val Lieu Gly Glin Ile Ile Ala Lys Glin Arg Gly Lieu Pro 660 665 67 O Ala Glu Lys Gly Glu Glu Met Wall Lys His Met Arg Ser Ser Gly Ser 675 680 685 Tyr Glin Asp Asp Val Trp Ser 69 O. 695
<210 SEQ ID NO 9 &2 11s LENGTH 695 &212> TYPE PRT <213> ORGANISM: Aspergillus ochraceus <400 SEQUENCE: 9 Met Ala Glin Lieu. Asp Thr Lieu. Asp Lieu Val Val Lieu Val Ala Lieu Lieu 1 5 10 15 US 2005/0003473 A1 Jan. 6, 2005 52
-continued
Val Gly Ser Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp Ala Val Ala 2O 25 30 Lys Asp Pro Tyr Ala Ser Ala Gly Pro Ala Met Asn Gly Gly Ala Lys 35 40 45 Ala Gly Lys Thr Arg Asp Ile Val Glin Lys Met Asp Glu Thr Gly Lys 50 55 60 Asn Cys Val Ile Phe Tyr Gly Ser Glin Thr Gly. Thr Ala Glu Asp Tyr 65 70 75 8O Ala Ser Arg Lieu Ala Lys Glu Gly Ser Glin Arg Phe Gly Lieu Lys Thr 85 90 95 Met Val Ala Asp Leu Glu Asp Tyr Asp Tyr Glu Asn Lieu Glu Lys Phe 100 105 110 Pro Glu Asp Llys Val Val Phe Phe Val Leu Ala Thr Tyr Gly Glu Gly 115 120 125 Glu Pro Thr Asp Asn Ala Val Glu Phe Tyr Glin Phe Val Thr Gly Glu 130 135 1 4 0 Asp Ala Ala Phe Glu Ser Gly Ala Thr Ala Asp Asp Llys Pro Leu Ser 145 15 O 155 160 Ser Leu Lys Tyr Val Thr Phe Gly Leu Gly Asn Asn Thr Tyr Glu His 1.65 170 175 Tyr Asn Ala Met Val Arg Asn Val Asp Ala Ala Lieu. Thir Lys Phe Gly 18O 185 19 O Ala Glin Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr 195 200 2O5 Met Glu Glu Asp Phe Lieu Ala Trp Lys Glu Pro Met Trp Ala Ala Lieu 210 215 220 Ser Glu Ala Met Asn Lieu Glin Glu Arg Asp Ala Val Tyr Glu Pro Val 225 230 235 240 Phe Asn Val Thr Glu Asp Glu Ser Lieu Ser Pro Glu Asp Glu Asn. Wal 245 250 255 Tyr Lieu Gly Glu Pro Thr Glin Gly His Leu Glin Gly Glu Pro Lys Gly 260 265 27 O Pro Tyr Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ser Glu Ser Arg 275 280 285 Glu Lieu Phe Asn. Wall Lys Asp Arg Asn. Cys Lieu. His Met Glu Ile Ser 29 O 295 3OO Ile Ala Gly Ser Asn Leu Thr Tyr Glin Thr Gly Asp His Ile Ala Val 305 310 315 320 Trp Pro Thr Asn Ala Gly Ser Glu Val Asp Arg Phe Leu Glin Ala Phe 325 330 335 Gly Lieu Glu Gly Lys Arg His Ser Val Ile Asn. Ile Lys Gly Ile Asp 340 345 35 O Val Thr Ala Lys Val Pro Ile Pro Thr Pro Thir Thr Tyr Asp Ala Ala 355 360 365 Val Arg Tyr Tyr Leu Glu Val Cys Ala Pro Val Ser Arg Glin Phe Val 370 375 38O Ser Thr Lieu Ala Ala Phe Ala Pro Asp Glu Ala Thr Lys Ala Glu Ile 385 390 395 400 Val Arg Lieu Gly Gly Asp Lys Asp Tyr Phe His Glu Lys Ile Thr Asn 405 410 415 US 2005/0003473 A1 Jan. 6, 2005 53
-continued Arg Cys Phe Asn. Ile Ala Glin Ala Lieu Glin Ser Ile Thr Ser Lys Pro 420 425 43 O Phe Thr Ala Val Pro Phe Ser Leu Leu Ile Glu Gly Ile Thr Lys Leu 435 4 40 4 45 Gln Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu Val Glin Lys Asp 450 455 460 Lys Ile Ser Ile Thr Ala Val Val Glu Ser Val Arg Leu Pro Gly Glu 465 470 475 480 Glu His Ile Val Lys Gly Val Thir Thr Asn Tyr Lieu Lleu Ala Lieu Lys 485 490 495 Glu Lys Glin Asn Gly Glu Pro Ser Pro Asp Pro His Gly Leu Thr Tyr 5 OO 505 51O. Ser Ile Thr Gly Pro Arg Asn Lys Tyr Asp Gly Ile His Val Pro Val 515 52O 525 His Val Arg His Ser Asn. Phe Lys Lieu Pro Ser Asp Pro Ser Arg Pro 530 535 540 Val Ile Met Val Gly Pro Gly. Thr Gly Val Ala Pro Phe Arg Gly Phe 545 550 555 560 Ile Glin Glu Arg Ala Ala Lieu Ala Ala Lys Gly Glu Lys Val Gly Thr 565 570 575 Thr Lieu Lieu Phe Phe Gly Cys Arg Lys Ser Asp Glu Asp Phe Leu Tyr 58O 585 59 O Lys Asp Glu Trip Lys Thr Phe Glin Glu Gln Leu Gly Asp Ser Lieu Lys 595 600 605 Ile Ile Thr Ala Phe Ser Arg Glu Ser Ala Glu Lys Val Tyr Val Glin 610 615 62O His Arg Lieu Arg Glu His Ala Glu Lieu Val Ser Asp Lieu Lleu Lys Glin 625 630 635 640 Lys Ala Thr Phe Tyr Val Cys Gly Asp Ala Ala Asn Met Ala Arg Glu 645 650 655 Val Asn Lieu Val Lieu Gly Glin Ile Ile Ala Lys Glin Arg Gly Lieu Pro 660 665 67 O Ala Glu Lys Gly Glu Glu Met Wall Lys His Met Arg Ser Ser Gly Ser 675 680 685 Tyr Glin Asp Asp Val Trp Ser 69 O. 695
<210> SEQ ID NO 10 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Homo sapiens primer H. oxred 1A <400 SEQUENCE: 10 gatcggat.cc aatatgg gag acticccacgt ggacac 36
<210> SEQ ID NO 11 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: Homo sapiens primer H. oxred 1B <400 SEQUENCE: 11 gatcggat.cc aatatgg gag acticccacgt ggacac 36
<210> SEQ ID NO 12 US 2005/0003473 A1 Jan. 6, 2005 54
-continued
<211& LENGTH 22 &212> TYPE DNA <213> ORGANISM homo sapiens primer H. oxred 2A <400 SEQUENCE: 12 citctgctdtc gtcaaccago to 22
<210> SEQ ID NO 13 &2 11s LENGTH 35 &212> TYPE DNA <213> ORGANISM: homo sapiens primer H. oxred2B <400 SEQUENCE: 13 gatcggtacc ttagcticcac acgtocaggg agtag 35
<210> SEQ ID NO 14 &2 11s LENGTH 2.0 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus primer A. oxred-for 1 &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (6) . . (6) <223> OTHER INFORMATION: n = Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (6) . . (6) <223> OTHER INFORMATION: n is a c, g, or t &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (9) ... (9) <223> OTHER INFORMATION: n = Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (9) ... (9) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 14 gacgging Cng gtacaatgga 20
<210 SEQ ID NO 15 &2 11s LENGTH 26 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev1. &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (4) ... (4) <223> OTHER INFORMATION: n = Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (4) ... (4) <223> OTHER INFORMATION: n is a c, g, or t &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (10) . . (10) <223> OTHER INFORMATION: n = Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (10) . . (10) <223> OTHER INFORMATION: n is a c, g, or t &220s FEATURE <221 NAME/KEY: modified base <222> LOCATION: (16) . . (16) <223> OTHER INFORMATION: n = Inosine &220s FEATURE <221 NAME/KEY: misc feature <222> LOCATION: (16) . . (16) <223> OTHER INFORMATION: n is a c, g, or t <400 SEQUENCE: 15 US 2005/0003473 A1 Jan. 6, 2005 55
-continued ttan gaccan acatcntcct ggtagc 26
<210> SEQ ID NO 16 &2 11s LENGTH 2.8 &212> TYPE DNA <213> ORGANISM: eschericia coli Primer pSport-for 1 <400 SEQUENCE: 16 caagctictaa tacgacticac tataggga 28
<210 SEQ ID NO 17 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev2 <400 SEQUENCE: 17 caggaaccga to gaccitcgg aa 22
<210> SEQ ID NO 18 &2 11s LENGTH 25 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev3 <400 SEQUENCE: 18 gtoaccctica ccagoaga.gc caatg 25
<210 SEQ ID NO 19 &2 11s LENGTH 2.8 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev4 <400 SEQUENCE: 19 ccacattgcg aaccatagog ttgtagtg 28
<210> SEQ ID NO 20 &2 11s LENGTH 34 &212> TYPE DNA <213> ORGANISM: eschericia coli Primer pSport-for-2 <400 SEQUENCE: 20 gccaagcticit aatacgactic actataggga aagc 34
<210> SEQ ID NO 21 &2 11s LENGTH 27 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-for-2 <400 SEQUENCE: 21 gtogacatgg cqcaactcga tactcitc 27
<210> SEQ ID NO 22 &2 11s LENGTH: 31 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev5 <400 SEQUENCE: 22 citcgagittag gaccagacat cqtcc togta g 31
<210> SEQ ID NO 23 <211& LENGTH 24 &212> TYPE DNA US 2005/0003473 A1 Jan. 6, 2005 56
-continued <213> ORGANISM: aspergillus ochraceus Primer A. oxred-for 3 <400 SEQUENCE: 23 ggat.ccct cq cqacct gtga totat 24
<210> SEQ ID NO 24 &2 11s LENGTH 38 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-for 4 <400 SEQUENCE: 24 cgaagattitc ttgtacaagg atgaatggaa gacttittc 38
<210> SEQ ID NO 25 &2 11s LENGTH 36 &212> TYPE DNA <213> ORGANISM: aspergillus ochraceus Primer A. oxred-rev6 <400 SEQUENCE: 25 citgaaaagttc titccattcat cottgtacaa gaaatc 36
<210> SEQ ID NO 26 &2 11s LENGTH 18 &212> TYPE PRT <213> ORGANISM: aspergillus ochraceus 11aoH peptide 1 <400> SEQUENCE: 26 Ala Ala Ala Tyr Trp Lieu Ala Thr Lieu Glin Pro Ser Asp Leu Pro Glu 1 5 10 15
Teu Asn
<210 SEQ ID NO 27 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM: aspergillus ochraceus 11aoH peptide 2 <400 SEQUENCE: 27 Cys Arg Glin Ile Leu Thr Pro Tyr Ile His Lys Arg Lys Ser Lieu Lys 1 5 10 15 Gly. Thir Thr Asp 2O
<210> SEQ ID NO 28 <211& LENGTH 21 &212> TYPE PRT <213> ORGANISM: aspergillus ochraceus 11aoH peptide 3 <400 SEQUENCE: 28 His Met Gly Phe Gly His Gly Val His Ala Cys Pro Gly Arg Phe Phe 1 5 10 15
Ala Ser Asn. Glu Ile 2O
<210 SEQ ID NO 29 &2 11s LENGTH 2.0 &212> TYPE PRT <213> ORGANISM homo sapiens oxir peptide 1 <400 SEQUENCE: 29 Cys Thr Tyr Trp Ala Val Ala Lys Asp Pro Tyr Ala Ser Ala Gly Pro US 2005/0003473 A1 Jan. 6, 2005 57
-continued
10 15 Ala Met Asn Gly
SEQ ID NO 30 LENGTH 526 TYPE PRT ORGANISM: gibberella fujikuroi CAA75565
<400 SEQUENCE: 30
Met Ala Asn His Ser Ser Ser Tyr Tyr His Glu Phe Lys Asp His 1 5 10 15
Ser His Thr Wall Teu Thr Teu Met Ser Glu Pro Wall Ile Telu Pro 25 30
Ser Telu Ile Telu Gly Thr Cys Ala Wall Telu Teu Ile Glin Trp Telu 35 40 45
Pro Glin Pro Teu Ile Met Wall Asn Gly Arg Lys Phe Gly Glu Telu 50 55 60
Ser Asn Wall Arg Ala Lys Arg Asp Phe Thr Phe Gly Ala Glin Telu 65 70 75
Teu Glu Gly Teu Met Ser Pro Asp Pro Phe Ile Met 85 90 95
Gly Asp Wall Gly Glu Lel His Ile Telu Pro Pro Ala Glu 100 105 110
Wall Arg Asn Asn Glu Teu Ser Phe Thr Met Ala Ala Phe Trp 115 120 125
Phe Tyr Ala His Teu Gly Phe Glu Gly Phe Arg Glu Gly Thr Asn 130 135 1 4 0
Glu Ser His Ile Met Lys Teu Wall Ala Arg His Glin Teu Thr His Glin 145 15 O 155 160
Teu Thr Telu Wall Thr Gly Ala Wall Ser Glu Glu Ala Telu Wall Telu 1.65 170 175
Asp Wall Tyr Thr Asp Ser Pro Glu Trp His Asp Ile Thr Ala Lys 18O 185 19 O
Asp Ala Asn Met Lys Teu Met Ala Arg Ile Thr Ser Arg Wall Phe Telu 195 200
Gly Lys Glu Met Cys Arg Asn Pro Glin Trp Teu Arg Ile Thr Ser Thr 210 215 220
Tyr Ala Wall Ile Ala Phe Wall Glu Glu Teu Arg Telu Trp Pro 225 230 235 240
Ser Trp Telu Pro Wall Wall Trp Phe Met Pro His Thr Glin 245 250 255
Ser Arg Ala Telu Wall Glin Glu Arg Asp Teu Ile Asn Pro Telu Telu 260 265 27 O
Glu Arg Arg Arg Glu Glu Lys Glu Ala Glu Arg Thr Gly Glu Lys 275 285
Wall Thr Asn Asp Ala Wall Trp Telu Asp Asp Teu Ala Arg Glu 29 O 295
Lys Gly Wall Gly Tyr Asp Pro Ala Glin Teu Ser Telu Ser Wall 305 310 315 320
Ala Ala Telu His Ser Thr Thr Phe Phe Thr Glin Wall Met Phe Asp 325 330 335 US 2005/0003473 A1 Jan. 6, 2005 58
-continued
Ile Ala Glin Asn Pro Glu Teu Ile Glu Pro Teu Arg Glu Glu Ile Ile 340 345 35 O
Ala Wall Telu Gly Lys Glin Gly Trp Ser Lys Asn Ser Teu Tyr Asn Telu 355 360 365
Telu Met Asp Ser Wall Teu Lys Glu Ser Glin Arg Teu Pro Ile 370 375
Ala Ile Ala Ser Met Arg Arg Phe Thr Thr His Asn Wall Telu Ser 385 390 395 400
Asp Gly Wall Ile Teu Pro Lys Asn Lys Telu Thr Teu Wall Ser Ala His 405 410 415
Glin His Trp Asp Pro Glu Tyr Tyr Lys Asp Pro Teu Phe Asp Gly 420 425 43 O
Arg Phe Phe Asn Met Arg Arg Glu Pro Gly Lys Glu Ser Ala 435 4 40 4 45
Glin Telu Wall Ser Ala Thr Pro Asp His Met Gly Phe Gly Tyr Gly Telu 450 455 460
His Ala Cys Pro Gly Arg Phe Phe Ala Ser Glu Glu Ile Ile Ala 465 470 475 480
Teu Ser His Ile Teu Teu Lys Tyr Phe Pro Wall Glu Gly Ser 485 490 495
Ser Met Glu Pro Lys Tyr Gly Telu Asn Met Asn Ala Asn Pro Thr 5 OO 505 51O.
Ala Lys Telu Ser Wall Arg Arg Lys Glu Glu Ile Ala Ile 515 52O 525
SEQ ID NO 31 LENGTH 514 TYPE PRT ORGANISM: neurospora crassa CAB91316
<400 SEQUENCE: 31
Met Glu Arg Lieu Asp Ile Lys Ser Ile Thr Asp Pro Ser Ala Thr Pro 1 5 10 15
Phe Ser Tyr Telu Wall Thr Ala Phe Telu Telu Ala Wall Wall Wall Ser 25 30
Teu Glin Gly Pro Phe Pro Lys Asn Ile His Teu Asn Pro 35 40 45
Gly Pro Telu Glu Phe Ser Asp Thr Pro Lys Glu Phe Wall 50 55 60
Gly Ser Arg Glin Met Teu Ala Asn Trp Phe Lys Ala Asn Pro Asn 65 70 75
Pro Arg Wall Ile Ser Asp Phe Gly Glu Ala Ile Wall Telu Pro Pro 85 90 95
Arg Met Ala Asn Glu Ile Lys Asn Asp Asp Arg Teu Ser Phe Thr Arg 100 105 110
Trp Thr Tyr Lys Ala Phe His Gly His Telu Pro Gly Phe Glu Gly Phe 115 120 125
Gly Glu Ala Ser Glu Ser His Ile Wall Glin Glu Wall Ile Met Arg 130 135 1 4 0
Asp Telu Thr Lys Tyr Teu Asn Lys Wall Thr Glu Pro Teu Ala Glin Glu 145 15 O 155 160
Thr Ser Met Ala Met Glu Ala Asn Telu Pro Ala Ala Asn Gly Glu 1.65 170 175 US 2005/0003473 A1 Jan. 6, 2005 59
-continued
Trp Ser Thir Ile Asn Lieu Arg Ser Lys Ile Leu Pro Ile Val Ala Arg 18O 185 19 O Ile Ser Ser Arg Val Phe Leu Gly Glu Glu Lieu. Cys Arg Asn. Glu Glu 195 200 2O5 Trp Leu Lys Val Thr Glin Gln Tyr Thr Ile Asp Gly Phe Gly Ala Ala 210 215 220 Glu Asp Leu Arg Lieu Trp Pro Ala Ala Lieu Arg Pro Ile Val His Trp 225 230 235 240 Phe Leu Pro Ser Cys Glin Arg Ala Arg Ala Asp Val Arg Val Ala Arg 245 250 255 Ser Ile Leu Asp Pro Val Lieu Lys Lys Arg Arg Glin Glu Lys Ala Ala 260 265 27 O Asn Gly Gly Lys Ala Glu His Asp Asp Ala Ile Glu Trp Phe Glu Arg 275 280 285 Thr Ala Lys Gly Lys Tyr Tyr Asp Pro Ala Val Ala Glin Leu Val Lieu 29 O 295 3OO Ser Leu Val Ala Ile His Thr Thr Ser Asp Leu Thr Cys Glin Val Met 305 310 315 320 Thr Asn Leu Met Gln Asn Pro Glu Phe Ile Ala Pro Leu Arg Glu Glu 325 330 335 Met Ile Glin Val Leu Ser Glu Gly Gly Trp Llys Lys Thr Ser Leu Tyr 340 345 35 O Asn Met Lys Lieu Lleu. Asp Ser Val Ile Lys Glu Ser Glin Arg Val Lys 355 360 365 Pro Thr Gly Val Ala Ser Met Arg Arg Tyr Ala Glu Lys Asp Val Thr 370 375 38O Leu Ser Asp Gly Thr Phe Ile Pro Lys Gly Gly Phe Val Ala Val Ser 385 390 395 400 Ala His Asp Met Trp Asn. Ser Glu Val Tyr Glu Glin Ala Glu Lys Trp 405 410 415 Asp Gly Arg Arg Phe Lieu Arg Met Arg Glu Thr Pro Gly Ala Gly Lys 420 425 43 O Glu Asin Val Ala Gln Leu Val Ser Thr Ala Pro Glu His Leu Gly Phe 435 4 40 4 45 Gly His Gly Glin His Ala Cys Pro Gly Arg Phe Phe Ala Ala Asn. Glu 450 455 460 Ile Lys Ile Ala Leu Val His Lieu Lleu Lieu. Asn Tyr Glu Trp Arg Lieu 465 470 475 480 Pro Glu Gly Ser Asp Pro Lys Ile Arg Thr Phe Gly Phe Ser Met Gly 485 490 495 Val Asp Pro Ser Lieu Lys Val Glu Tyr Lys Gly Arg Glin Pro Glu Ile 5 OO 505 51O.
Glu Lieu
<210> SEQ ID NO 32 &2 11s LENGTH 495 &212> TYPE PRT <213> ORGANISM: Catharanthus roseus CAB56503
<400 SEQUENCE: 32 Leu Leu Phe Cys Phe Ile Leu Ser Lys Thr Thr Lys Lys Phe Gly Glin 1 5 10 15 US 2005/0003473 A1 Jan. 6, 2005 60
-continued
Asn Ser Glin Tyr Ser Asn His Asp Glu Leu Pro Pro Gly Pro Pro Glin 2O 25 30 Ile Pro Ile Leu Gly Asn Ala His Glin Leu Ser Gly Gly His Thr His 35 40 45 His Ile Leu Arg Asp Leu Ala Lys Lys Tyr Gly Pro Leu Met His Lieu 50 55 60 Lys Ile Gly Glu Val Ser Thr Ile Val Ala Ser Ser Pro Glin Ile Ala 65 70 75 8O Glu Glu Ile Phe Arg Thr His Asp Ile Leu Phe Ala Asp Arg Pro Ser 85 90 95 Asn Leu Glu Ser Phe Lys Ile Val Ser Tyr Asp Phe Ser Asp Met Val 100 105 110 Val Ser Pro Tyr Gly Asn Tyr Trp Arg Gln Leu Arg Lys Ile Ser Met 115 120 125 Met Glu Lieu Lleu Ser Glin Lys Ser Val Glin Ser Phe Arg Ser Ile Arg 130 135 1 4 0 Glu Glu Glu Val Lieu. Asn. Phe Ile Lys Ser Ile Gly Ser Lys Glu Gly 145 15 O 155 160 Thr Arg Ile Asn Lieu Ser Lys Glu Ile Ser Lieu Lleu. Ile Tyr Gly Ile 1.65 170 175 Thir Thr Arg Ala Ala Phe Gly Glu Lys Asn Lys Asn Thr Glu Glu Phe 18O 185 19 O Ile Arg Lieu Lieu. Asp Glin Lieu. Thir Lys Ala Wall Ala Glu Pro Asn. Ile 195 200 2O5 Ala Asp Met Phe Pro Ser Leu Lys Phe Leu Gln Leu Ile Ser Thr Ser 210 215 220 Lys Tyr Lys Ile Glu Lys Ile His Lys Glin Phe Asp Val Ile Val Glu 225 230 235 240 Thir Ile Leu Lys Gly His Lys Glu Lys Ile Asn Lys Pro Leu Ser Glin 245 250 255 Glu Asn Gly Glu Lys Lys Glu Asp Leu Val Asp Val Lieu Lieu. Asn. Ile 260 265 27 O Glin Arg Arg Asn Asp Phe Glu Ala Pro Leu Gly Asp Lys Asn. Ile Lys 275 280 285 Ala Ile Ile Phe Asn Ile Phe Ser Ala Gly Thr Glu Thir Ser Ser Thr 29 O 295 3OO Thr Val Asp Trp Ala Met Cys Glu Met Ile Lys Asn Pro Thr Val Met 305 310 315 320 Lys Lys Ala Glin Glu Glu Val Arg Llys Val Phe Asn. Glu Glu Gly Asn 325 330 335 Val Asp Glu Thir Lys Lieu. His Glin Lieu Lys Tyr Lieu Glin Ala Val Ile 340 345 35 O Lys Glu Thir Lieu Arg Lieu. His Pro Pro Val Pro Leu Lleu Lleu Pro Arg 355 360 365 Glu Cys Arg Glu Glin Cys Lys Ile Lys Gly Tyr Thr Ile Pro Ser Lys 370 375 38O Ser Arg Val Ile Val Asn Ala Trp Ala Ile Gly Arg Asp Pro Asn Tyr 385 390 395 400 Trp Ile Glu Pro Glu Lys Phe Asn Pro Asp Arg Phe Lieu Glu Ser Lys 405 410 415 US 2005/0003473 A1 Jan. 6, 2005 61
-continued Val Asp Phe Lys Gly Asn Ser Phe Glu Tyr Leu Pro Phe Gly Gly Gly 420 425 43 O Arg Arg Ile Cys Pro Gly Ile Thr Phe Ala Lieu Ala Asn. Ile Glu Lieu 435 4 40 4 45 Pro Leu Ala Gln Leu Leu Phe His Phe Asp Trp Gln Ser Asn Thr Glu 450 455 460 Lys Lieu. Asn Met Lys Glu Ser Arg Gly Val Thr Val Arg Arg Glu Asp 465 470 475 480 Asp Leu Tyr Leu Thr Pro Val Asin Phe Ser Ser Ser Ser Pro Ala 485 490 495
<210 SEQ ID NO 33 &2 11s LENGTH 510 &212> TYPE PRT <213> ORGANISM: glycine max AAB94588 <400 SEQUENCE: 33 Met Val Met Glu Lieu. His Asn His Thr Pro Phe Ser Ile Tyr Phe Ile 1 5 10 15 Thr Ser Ile Leu Phe Ile Phe Phe Val Phe Phe Lys Leu Val Glin Arg 2O 25 30 Ser Asp Ser Lys Thr Ser Ser Thr Cys Lys Leu Pro Pro Gly Pro Arg 35 40 45 Thr Leu Pro Leu Ile Gly Asn. Ile His Glin Ile Val Gly Ser Leu Pro 50 55 60 Val His Tyr Tyr Lieu Lys Asn Lieu Ala Asp Llys Tyr Gly Pro Leu Met 65 70 75 8O His Leu Lys Leu Gly Glu Val Ser Asn Ile Ile Val Thr Ser Pro Glu 85 90 95 Met Ala Glin Glu Ile Met Lys Thr His Asp Lieu. Asn. Phe Ser Asp Arg 100 105 110 Pro Asp Phe Val Leu Ser Arg Ile Val Ser Tyr Asn Gly Ser Gly Ile 115 120 125 Val Phe Ser Glin His Gly Asp Tyr Trp Arg Glin Leu Arg Lys Ile Cys 130 135 1 4 0 Thr Val Glu Leu Leu Thr Ala Lys Arg Val Glin Ser Phe Arg Ser Ile 145 15 O 155 160 Arg Glu Glu Glu Val Ala Glu Lieu Val Lys Lys Ile Ala Ala Thr Ala 1.65 170 175 Ser Glu Glu Gly Gly Ser Ile Phe Asn Leu Thr Glin Ser Ile Tyr Ser 18O 185 19 O Met Thr Phe Gly Ile Ala Ala Arg Ala Ala Phe Gly Lys Lys Ser Arg 195 200 2O5 Tyr Glin Glin Val Phe Ile Ser Asn Met His Lys Gln Leu Met Leu Leu 210 215 220 Gly Gly Phe Ser Val Ala Asp Leu Tyr Pro Ser Ser Arg Val Phe Glin 225 230 235 240 Met Met Gly Ala Thr Gly Lys Lieu Glu Lys Wal His Arg Val Thr Asp 245 250 255 Arg Val Lieu Glin Asp Ile Ile Asp Glu His Lys Asn Arg Asn Arg Ser 260 265 27 O Ser Glu Glu Arg Glu Ala Val Glu Asp Lieu Val Asp Wall Leu Lleu Lys 275 280 285 US 2005/0003473 A1 Jan. 6, 2005 62
-continued
Phe Glin Lys Glu Ser Glu Phe Arg Lieu. Thir Asp Asp Asn. Ile Lys Ala 29 O 295 3OO Val Ile Glin Asp Ile Phe Ile Gly Gly Gly Glu Thir Ser Ser Ser Val 305 310 315 320 Val Glu Trp Gly Met Ser Glu Leu Ile Arg Asn Pro Arg Val Met Glu 325 330 335 Glu Ala Glin Ala Glu Val Arg Arg Val Tyr Asp Ser Lys Gly Tyr Val 340 345 35 O Asp Glu Thr Glu Lieu. His Glin Lieu. Ile Tyr Lieu Lys Ser Ile Ile Lys 355 360 365 Glu Thr Met Arg Leu. His Pro Pro Val Pro Leu Leu Val Pro Arg Val 370 375 38O Ser Arg Glu Arg Cys Glin Ile Asin Gly Tyr Glu Ile Pro Ser Lys Thr 385 390 395 400 Arg Ile Ile Ile Asn Ala Trp Ala Ile Gly Arg Asn Pro Llys Tyr Trp 405 410 415 Gly Glu Thr Glu Ser Phe Lys Pro Glu Arg Phe Leu Asn Ser Ser Ile 420 425 43 O Asp Phe Arg Gly Thr Asp Phe Glu Phe Ile Pro Phe Gly Ala Gly Arg 435 4 40 4 45 Arg Ile Cys Pro Gly Ile Thr Phe Ala Ile Pro Asn Ile Glu Leu Pro 450 455 460 Leu Ala Glin Leu Lleu Tyr His Phe Asp Trp Llys Lieu Pro Asn Lys Met 465 470 475 480 Lys Asn. Glu Glu Lieu. Asp Met Thr Glu Ser Asn Gly Ile Thr Lieu Arg 485 490 495 Arg Glin Asn Asp Lieu. Cys Lieu. Ile Pro Ile Thr Arg Lieu Pro 5 OO 505 51O.
<210> SEQ ID NO 34 &2 11s LENGTH 524 &212> TYPE PRT <213> ORGANISM: Gibberella fujikuroi CAA755 66 <400 SEQUENCE: 34 Met Ser Ile Phe Asn Met Ile Thr Ser Tyr Ala Gly Ser Gln Leu Leu 1 5 10 15 Pro Phe Tyr Ile Ala Ile Phe Val Phe Thr Leu Val Pro Trp Ala Ile 2O 25 30 Arg Phe Ser Trp Leu Glu Lieu Arg Lys Gly Ser Val Val Pro Leu Ala 35 40 45 Asn Pro Pro Asp Ser Leu Phe Gly Thr Gly Lys Thr Arg Arg Ser Phe 50 55 60 Wall Lys Lieu Ser Arg Glu Ile Leu Ala Lys Ala Arg Ser Lieu Phe Pro 65 70 75 8O Asn Glu Pro Phe Arg Lieu. Ile Thr Asp Trp Gly Glu Val Lieu. Ile Leu 85 90 95 Pro Pro Asp Phe Ala Asp Glu Ile Arg Asn Asp Pro Arg Lieu Ser Phe 100 105 110 Ser Lys Ala Ala Met Glin Asp Asn His Ala Gly Ile Pro Gly Phe Glu 115 120 125 Thr Val Ala Lieu Val Gly Arg Glu Asp Gln Lieu. Ile Glin Lys Val Ala US 2005/0003473 A1 Jan. 6, 2005 63
-continued
130 135 1 4 0 Arg Lys Glin Lieu. Thir Lys His Leu Ser Ala Val Ile Glu Pro Leu Ser 145 15 O 155 160 Arg Glu Ser Thr Leu Ala Val Ser Leu Asin Phe Gly Glu Thir Thr Glu 1.65 170 175 Trp Arg Ala Ile Arg Lieu Lys Pro Ala Ile Leu Asp Ile Ile Ala Arg 18O 185 19 O Ile Ser Ser Arg Ile Tyr Lieu Gly Asp Gln Lieu. Cys Arg Asn. Glu Ala 195 200 2O5 Trp Leu Lys Ile Thr Lys Thr Tyr Thr Thr Asn Phe Tyr Thr Ala Ser 210 215 220 Thr Asn Leu Arg Met Phe Pro Arg Ser Ile Arg Pro Leu Ala His Trp 225 230 235 240 Phe Leu Pro Glu Cys Arg Lys Lieu Arg Glin Glu Arg Lys Asp Ala Ile 245 250 255 Gly Ile Ile Thr Pro Lieu. Ile Glu Arg Arg Arg Glu Lieu Arg Arg Ala 260 265 27 O Ala Ile Ala Ala Gly Glin Pro Leu Pro Val Phe His Asp Ala Ile Asp 275 280 285 Trp Ser Glu Glin Glu Ala Glu Ala Ala Gly Thr Gly Ala Ser Phe Asp 29 O 295 3OO
Pro Wall Ile Phe Glin Leu Thir Leu Ser Leu Leu Ala Ile His Thir Thr 305 310 315 320 Tyr Asp Lieu Lieu Glin Glin Thr Met Ile Asp Leu Gly Arg His Pro Glu 325 330 335 Tyr Ile Glu Pro Leu Arg Glin Glu Val Val Glin Leu Lleu Arg Glu Glu 340 345 35 O Gly Trp Llys Lys Thr Thr Lieu Phe Lys Met Lys Lieu Lieu. Asp Ser Ala 355 360 365 Ile Lys Glu Ser Glin Arg Met Lys Pro Gly Ser Ile Val Thr Met Arg 370 375 38O Arg Tyr Val Thr Glu Asp Ile Thr Leu Ser Ser Gly Leu Thr Leu Lys 385 390 395 400 Lys Gly. Thir Arg Lieu. Asn. Wall Asp Asn Arg Arg Lieu. Asp Asp Pro Lys 405 410 415 Ile Tyr Asp Asin Pro Glu Val Tyr Asn Pro Tyr Arg Phe Tyr Asp Met 420 425 43 O Arg Ser Glu Ala Gly Lys Asp His Gly Ala Glin Lieu Val Ser Thr Gly 435 4 40 4 45 Ser Asn His Met Gly Phe Gly His Gly Glin His Ser Cys Pro Gly Arg 450 455 460 Phe Phe Ala Ala Asn. Glu Ile Llys Val Ala Lieu. Cys His Ile Leu Val 465 470 475 480 Lys Tyr Asp Trp Lys Lieu. Cys Pro Asp Thr Glu Thir Lys Pro Asp Thr 485 490 495 Arg Gly Met Ile Ala Lys Ser Ser Pro Val Thr Asp Ile Lieu. Ile Lys 5 OO 505 51O. Arg Arg Glu Ser Val Glu Lieu. Asp Leu Glu Ala Ile 515 52O
<210 SEQ ID NO 35 US 2005/0003473 A1 Jan. 6, 2005 64
-continued
&2 11s LENGTH 528 &212> TYPE PRT <213> ORGANISM: Aspergillus terreus AAD34552 <400 SEQUENCE: 35 Met Thr Val Asp Ala Leu Thr Glin Pro His His Leu Leu Ser Leu Ala 1 5 10 15 Trp Asin Asp Thr Glin Gln His Gly Ser Trp Phe Ala Pro Leu Val Thr 2O 25 30 Thir Ser Ala Gly Lieu Lieu. Cys Lieu Lleu Lleu Tyr Lieu. Cys Ser Ser Gly 35 40 45 Arg Arg Ser Asp Leu Pro Val Phe Asn Pro Llys Thr Trp Trp Glu Lieu 50 55 60 Thir Thr Met Arg Ala Lys Arg Asp Phe Asp Ala Asn Ala Pro Ser Trp 65 70 75 8O Ile Glu Ser Trp Phe Ser Glin Asn Asp Llys Pro Ile Arg Phe Ile Val 85 90 95 Asp Ser Gly Tyr Cys Thr Ile Leu Pro Ser Ser Met Ala Asp Glu Phe 100 105 110 Arg Lys Met Lys Glu Lieu. Cys Met Tyr Lys Phe Leu Gly Thr Asp Phe 115 120 125 His Ser His Leu Pro Gly Phe Asp Gly Phe Lys Glu Val Thr Arg Asp 130 135 1 4 0 Ala His Leu Ile Thr Lys Val Val Met Asin Glin Phe Gln Thr Glin Ala 145 15 O 155 160 Pro Llys Tyr Val Lys Pro Leu Ala Asn. Glu Ala Ser Gly Ile Ile Thr 1.65 170 175 Asp Ile Phe Gly Asp Ser Asn Glu Trp His Thr Val Pro Val Tyr Asn 18O 185 19 O Gln Cys Leu Asp Leu Val Thr Arg Thr Val Thr Phe Ile Met Val Gly 195 200 2O5 Ser Lys Lieu Ala His Asn. Glu Glu Trp Lieu. Asp Ile Ala Lys His His 210 215 220 Ala Val Thr Met Ala Ile Glin Ala Arg Gln Leu Arg Leu Trp Pro Val 225 230 235 240 Ile Leu Arg Pro Leu Val His Trp Leu Glu Pro Glin Gly Ala Lys Lieu 245 250 255 Arg Ala Glin Val Arg Arg Ala Arg Glin Leu Lieu. Asp Pro Ile Ile Glin 260 265 27 O Glu Arg Arg Ala Glu Arg Asp Ala Cys Arg Ala Lys Gly Ile Glu Pro 275 280 285 Pro Arg Tyr Val Asp Ser Ile Glin Trp Phe Glu Asp Thr Ala Lys Gly 29 O 295 3OO Lys Trp Tyr Asp Ala Ala Gly Ala Glin Leu Ala Met Asp Phe Ala Gly 305 310 315 320 Ile Tyr Gly Thir Ser Asp Leu Lieu. Ile Gly Gly Lieu Val Asp Ile Val 325 330 335 Arg His Pro His Leu Lleu Glu Pro Leu Arg Asp Glu Ile Arg Thr Val 340 345 35 O Ile Gly Glin Gly Gly Trp Thr Pro Ala Ser Leu Tyr Lys Lieu Lys Lieu 355 360 365 Leu Asp Ser Cys Lieu Lys Glu Ser Glin Arg Val Lys Pro Val Glu Cys US 2005/0003473 A1 Jan. 6, 2005 65
-continued
370 375 38O Ala Thr Met Arg Ser Tyr Ala Leu Glin Asp Val Thr Phe Ser Asn Gly 385 390 395 400 Thr Phe Ile Pro Lys Gly Glu Lieu Val Ala Val Ala Ala Asp Arg Met 405 410 415 Ser Asn Pro Glu Val Trp Pro Glu Pro Ala Lys Tyr Asp Pro Tyr Arg 420 425 43 O Tyr Met Arg Lieu Arg Glu Asp Pro Ala Lys Ala Phe Ser Ala Glin Lieu 435 4 40 4 45 Glu Asn. Thir Asn Gly Asp His Ile Gly Phe Gly Trp His Pro Arg Ala 450 455 460 Cys Pro Gly Arg Phe Phe Ala Ser Lys Glu Ile Lys Met Met Leu Ala 465 470 475 480 Tyr Lieu Lieu. Ile Arg Tyr Asp Trp Llys Val Val Pro Asp Glu Pro Leu 485 490 495 Gln Tyr Tyr Arg His Ser Phe Ser Val Arg Ile His Pro Thr Thr Lys 5 OO 505 51O. Leu Met Met Arg Arg Arg Asp Glu Asp Ile Arg Lieu Pro Gly Ser Lieu 515 52O 525
<210 SEQ ID NO 36 &2 11s LENGTH 388 &212> TYPE PRT <213> ORGANISM: Gibberella fujikuroi CAA755 67 <400 SEQUENCE: 36 Met Lys Tyr Thr Thr Cys Gln Met Asin Ile Phe Pro Ser Leu Trp Ser 1 5 10 15 Met Lys Thr Ser Phe Arg Trp Pro Arg Thr Ser Lys Trp Ser Ser Val 2O 25 30 Ser Leu Tyr Asp Met Met Lieu Arg Thr Val Ala Leu Lleu Ser Gly Arg 35 40 45 Ala Phe Val Gly Lieu Pro Leu. Cys Arg Asp Glu Gly Trp Leu Glin Ala 50 55 60 Ser Ile Gly Tyr Thr Val Glin Cys Val Ser Ile Arg Asp Glin Leu Phe 65 70 75 8O Thir Trp Ser Pro Val Leu Arg Pro Ile Ile Gly Pro Phe Leu Pro Ser 85 90 95 Val Arg Ser Val Arg Arg His Leu Arg Phe Ala Ala Glu Ile Met Ala 100 105 110 Pro Lieu. Ile Ser Glin Ala Leu Glin Asp Glu Lys Glin His Arg Ala Asp 115 120 125 Thr Leu Leu Ala Asp Glin Thr Glu Gly Arg Gly Thr Phe Ile Ser Trp 130 135 1 4 0 Leu Lieu Arg His Leu Pro Glu Glu Lieu Arg Thr Pro Glu Glin Val Gly 145 15 O 155 160 Leu Asp Gln Met Leu Val Ser Phe Ala Ala Ile His Thr Thr Thr Met 1.65 170 175 Ala Leu Thir Lys Val Val Trp Glu Leu Val Lys Arg Pro Glu Tyr Ile 18O 185 19 O Glu Pro Leu Arg Thr Glu Met Glin Asp Val Phe Gly Pro Asp Ala Val 195 200 2O5 US 2005/0003473 A1 Jan. 6, 2005 66
-continued Ser Pro Asp Ile Cys Ile Asn Lys Glu Ala Leu Ser Arg Lieu. His Lys 210 215 220 Leu Asp Ser Phe Ile Arg Glu Val Glin Arg Trp Cys Pro Ser Thr Phe 225 230 235 240 Val Thr Pro Ser Arg Arg Val Met Lys Ser Met Thr Leu Ser Asn Gly 245 250 255 Ile Lys Lieu Glin Arg Gly Thr Ser Ile Ala Phe Pro Ala His Ala Ile 260 265 27 O His Met Ser Glu Glu Thr Pro Thr Phe Ser Pro Asp Phe Ser Ser Asp 275 280 285 Phe Glu Asn Pro Ser Pro Arg Ile Phe Asp Gly Phe Arg Tyr Leu Asn 29 O 295 3OO Leu Arg Ser Ile Lys Gly Glin Gly Ser Gln His Glin Ala Ala Thir Thr 305 310 315 320 Gly Pro Asp Tyr Lieu. Ile Phe Asn His Gly Lys His Ala Cys Pro Gly 325 330 335 Arg Phe Phe Ala Ile Ser Glu Ile Lys Met Ile Lieu. Ile Glu Lieu Lieu 340 345 35 O Ala Lys Tyr Asp Phe Arg Lieu Glu Asp Gly Lys Pro Gly Pro Glu Lieu 355 360 365 Met Arg Val Gly Thr Glu Thr Arg Lieu. Asp Thr Lys Ala Gly Lieu Glu 370 375 38O Met Arg Arg Arg 385
<210 SEQ ID NO 37 &2 11s LENGTH 525 &212> TYPE PRT <213> ORGANISM: Gibberella fujikuroi CAA76703 <400 SEQUENCE: 37 Met Ser Lys Ser Asn Ser Met Asn Ser Thr Ser His Glu Thr Leu Phe 1 5 10 15 Glin Glin Lieu Val Lieu Gly Lieu. Asp Arg Met Pro Leu Met Asp Wal His 2O 25 30 Trp Leu Ile Tyr Val Ala Phe Gly Ala Trp Leu Cys Ser Tyr Val Ile 35 40 45 His Val Leu Ser Ser Ser Ser Thr Val Lys Val Pro Val Val Gly Tyr 50 55 60 Arg Ser Val Phe Glu Pro Thir Trp Leu Leu Arg Leu Arg Phe Val Trp 65 70 75 8O Glu Gly Gly Ser Ile Ile Gly Glin Gly Tyr Asn Lys Phe Lys Asp Ser 85 90 95 Ile Phe Glin Val Arg Lys Leu Gly Thr Asp Ile Val Ile Ile Pro Pro 100 105 110 Asn Tyr Ile Asp Glu Val Arg Lys Lieu Ser Glin Asp Lys Thr Arg Ser 115 120 125 Val Glu Pro Phe Ile Asn Asp Phe Ala Gly Glin Tyr Thr Arg Gly Met 130 135 1 4 0 Val Phe Leu Glin Ser Asp Leu Glin Asn Arg Val Ile Glin Glin Arg Lieu 145 15 O 155 160 Thr Pro Llys Lieu Val Ser Lieu. Thir Lys Wal Met Lys Glu Glu Lieu. Asp 1.65 170 175 US 2005/0003473 A1 Jan. 6, 2005 67
-continued
Tyr Ala Lieu. Thir Lys Glu Met Pro Asp Met Lys Asn Asp Glu Trp Val 18O 185 19 O Glu Val Asp Ile Ser Ser Ile Met Val Arg Leu Ile Ser Arg Ile Ser 195 200 2O5 Ala Arg Val Phe Leu Gly Pro Glu His Cys Arg Asn Glin Glu Trp Lieu 210 215 220 Thir Thr Thr Ala Glu Tyr Ser Glu Ser Leu Phe Ile Thr Gly Phe Ile 225 230 235 240 Leu Arg Val Val Pro His Ile Leu Arg Pro Phe Ile Ala Pro Leu Leu 245 250 255 Pro Ser Tyr Arg Thr Lieu Lleu Arg Asn. Wal Ser Ser Gly Arg Arg Val 260 265 27 O Ile Gly Asp Ile Ile Arg Ser Glin Glin Gly Asp Gly Asn. Glu Asp Ile 275 280 285 Leu Ser Trp Met Arg Asp Ala Ala Thr Gly Glu Glu Lys Glin Ile Asp 29 O 295 3OO Asn. Ile Ala Glin Arg Met Lieu. Ile Leu Ser Lieu Ala Ser Ile His Thr 305 310 315 320 Thr Ala Met Thr Met Thr His Ala Met Tyr Asp Leu Cys Ala Cys Pro 325 330 335 Glu Tyr Ile Glu Pro Leu Arg Asp Glu Wall Lys Ser Val Val Gly Ala 340 345 35 O Ser Gly Trp Asp Lys Thr Ala Lieu. Asn Arg Phe His Lys Lieu. Asp Ser 355 360 365 Phe Leu Lys Glu Ser Glin Arg Phe Asin Pro Val Phe Leu Leu Thr Phe 370 375 38O Asn Arg Ile Tyr His Glin Ser Met Thr Leu Ser Asp Gly Thr Asn Ile 385 390 395 400 Pro Ser Gly Thr Arg Ile Ala Val Pro Ser His Ala Met Leu Glin Asp 405 410 415 Ser Ala His Val Pro Gly Pro Thr Pro Pro Thr Glu Phe Asp Gly Phe 420 425 43 O Arg Tyr Ser Lys Ile Arg Ser Asp Ser Asn Tyr Ala Glin Lys Tyr Lieu 435 4 40 4 45 Phe Ser Met Thr Asp Ser Ser Asn Met Ala Phe Gly Tyr Gly Lys Tyr 450 455 460 Ala Cys Pro Gly Arg Phe Tyr Ala Ser Asn. Glu Met Lys Lieu. Thir Lieu 465 470 475 480 Ala Ile Leu Lleu Lleu Glin Phe Glu Phe Lys Lieu Pro Asp Gly Lys Gly 485 490 495 Arg Pro Arg Asn. Ile Thir Ile Asp Ser Asp Met Ile Pro Asp Pro Arg 5 OO 505 51O. Ala Arg Lieu. Cys Val Arg Lys Arg Ser Lieu Arg Asp Glu 515 52O 525
<210 SEQ ID NO 38 &2 11s LENGTH 2.94 &212> TYPE PRT <213> ORGANISM: Fusarium oxysporum CAA57874 <400 SEQUENCE: 38 Met Ala Pro Met Leu Arg Pro Leu Val Tyr Arg Phe Ile Pro Glu Arg US 2005/0003473 A1 Jan. 6, 2005 68
-continued
1 5 10 15 Ala Arg Ile Lys Asp Gln Trp Thr Lys Gly Arg Lys Arg Val Met Ala 2O 25 30 Ser Met Arg Glu Arg Glin Glu Lys Gly Gly Asn Lieu Glu Asp Pro Pro 35 40 45 Thr Met Lieu. Asp His Leu Ser Asn Gly Arg Asn. Glu His Ile Ala Asp 50 55 60 Asp Val Glu Lieu Gln Lieu Lleu. His Gln Met Thr Lieu. Ile Ala Val Gly 65 70 75 8O Thr Val Thr Thr Phe Ser Ser Thr Thr Glin Ala Ile Tyr Asp Leu Val 85 90 95 Ala His Pro Glu Tyr Ile Thir Ile Leu Arg Glu Glu Val Glu Ser Val 100 105 110 Pro Arg Asp Pro Asn Gly Asn Phe Thr Lys Asp Ser Thr Val Ala Met 115 120 125 Asp Llys Lieu. Asp Ser Phe Leu Lys Glu Ser Glin Arg Phe Asn. Ser Pro 130 135 1 4 0 Asp Leu Ser Met Ser Asn Lieu Lys Asn Tyr Lys Lieu. Cys Glu Ser Lieu 145 15 O 155 160 Thr Gly. His Ser Asn Leu Pro Thr Arg Thr Ile Ala Asp Met Lys Leu 1.65 170 175 Pro Asp Gly Thr Phe Val Pro Lys Gly Thr Lys Leu Glu Ile Asn Thr 18O 185 19 O Cys Ser Ile His Lys Asp His Lys Lieu. Tyr Glu Asn Pro Glu Glin Phe 195 200 2O5 Asp Gly Lieu Arg Phe His Lys Trp Arg Lys Ala Pro Gly Lys Glu Lys 210 215 220 Arg Tyr Met Tyr Ser Ser Ser Gly Thr Asp Asp Leu Ser Trp Gly Phe 225 230 235 240 Gly Arg His Ala Cys Pro Gly Arg Tyr Lieu Ser Ala Ile Asn. Ile Lys 245 250 255 Lieu. Ile Met Ala Glu Lieu Lleu Met Asn Tyr Asp Ile Lys Lieu Pro Asp 260 265 27 O Gly Lieu Ser Arg Pro Lys Asn. Ile Glu Phe Glu Val Lieu Ala Ser Lieu 275 280 285 Asn Ala Cys Ala Asn Ala 29 O
<210 SEQ ID NO 39 &2 11s LENGTH 510 &212> TYPE PRT <213> ORGANISM: Caenorhabditis elegans CAA9 1268 <400 SEQUENCE: 39
Met Ala Leu Lleu. Ile Leu Ser Ser Leu Wall Ile Ser Ile Phe Thr Phe 1 5 10 15 Phe Ile Tyr Ile Ile Leu Ala Arg Arg Glu Arg Phe Lys Lieu Arg Glu 2O 25 30 Lys Ile Gly Lieu Ser Gly Pro Glu Pro His Trp Phe Lieu Gly Asn Lieu 35 40 45 Lys Glin Thr Ala Glu Arg Lys Glu Lys Lieu Gly Tyr Asp Asp Ala Asn 50 55 60 US 2005/0003473 A1 Jan. 6, 2005 69
-continued Arg Trp Phe Asin Glu Lieu. His Glu Gln Tyr Gly Glu Thr Phe Gly Ile 65 70 75 8O Tyr Tyr Gly Ser Glin Met Asn. Ile Val Ile Ser Asn. Glu Lys Asp Ile 85 90 95 Lys Glu Val Phe Ile Lys Asn. Phe Ser Asn. Phe Ser Asp Arg Ser Val 100 105 110 Pro Ser Ile Tyr Glu Ala Asn Glin Lieu. Thir Ala Ser Lieu Lleu Met Asn 115 120 125 Ser Tyr Ser Ser Gly Trp Lys His Thr Arg Ser Ala Ile Ala Pro Ile 130 135 1 4 0 Phe Ser Thr Gly Lys Met Lys Ala Met Glin Glu Thir Ile Asin Ser Lys 145 15 O 155 160 Val Asp Leu Phe Leu Asp Ile Leu Arg Glu Lys Ala Ser Ser Gly Glin 1.65 170 175 Lys Trp Asp Ile Tyr Asp Asp Phe Glin Gly Lieu. Thir Lieu. Asp Val Ile 18O 185 19 O Gly Lys Cys Ala Phe Ala Ile Asp Ser Asn. Cys Glin Arg Asp Arg Asn 195 200 2O5 Asp Val Phe Tyr His Pro Val Thr Val Lys Ile Thr Ile Asin Asn Phe 210 215 220 Thr Tyr Phe His Ser Ser Ser Pro Gly Thr Phe His Phe Leu Glu Ser 225 230 235 240 Thr Leu Glin Ile His Thr Thr Gly Arg Cys Arg Asn Ser Thr Cys Arg 245 250 255 Arg Thr Val Lys Cys Val Gly Phe Arg Glin Asp Lys Ala Lys Phe Cys 260 265 27 O Ser Asp Tyr Glu Arg Arg Arg Gly Gly Glu Gly Ser Asp Ser Val Asp 275 280 285 Leu Lleu Lys Lieu Lleu Lieu. Asn Arg Glu Asp Asp Lys Ser Lys Pro Met 29 O 295 3OO Thr Lys Glin Glu Val Ile Glu Asn. Cys Phe Ala Phe Lieu Lieu Ala Gly 305 310 315 320 Tyr Glu Thir Thr Ser Thr Ala Met Thr Tyr Cys Ser Tyr Leu Leu Ser 325 330 335 Lys Tyr Pro Asn Val Glin Gln Lys Lieu. Tyr Glu Glu Ile Met Glu Ala 340 345 35 O Lys Glu Asn Gly Gly Lieu. Thir Tyr Asp Ser Ile His Asn Met Lys Tyr 355 360 365 Leu Asp Cys Val Tyr Lys Glu Thr Leu Arg Phe Tyr Pro Pro His Phe 370 375 38O Ser Phe Ile Arg Arg Lieu. Cys Arg Glu Asp Ile Thr Ile Arg Gly Glin 385 390 395 400 Phe Tyr Pro Lys Gly Ala Ile Val Val Cys Leu Pro His Thr Val His 405 410 415 Arg Asn Pro Glu Asn Trp Asp Ser Pro Glu Glu Phe His Pro Glu Arg 420 425 43 O Phe Glu Asn Trp Glu Glu Lys Ser Ser Ser Leu Lys Trp Ile Pro Phe 435 4 40 4 45 Gly Val Gly Pro Arg Tyr Cys Val Gly Met Arg Phe Ala Glu Met Glu 450 455 460 Phe Lys Thr Thr Ile Val Lys Leu Leu Asp Thr Phe Glu Leu Lys Glin US 2005/0003473 A1 Jan. 6, 2005 70
-continued
465 470 475 480 Phe Glu Gly Glu Ala Asp Lieu. Ile Pro Asp Cys Asn Gly Val Ile Met 485 490 495 Arg Pro Asn Asp Pro Val Arg Lieu. His Lieu Lys Pro Arg Asn 5 OO 505 51O.
<210> SEQ ID NO 40 &2 11s LENGTH 691 &212> TYPE PRT <213> ORGANISM: saccahromyces cerevisiae P450 reductase <400 SEQUENCE: 40 Met Pro Phe Gly Ile Asp Asn Thr Asp Phe Thr Val Leu Ala Gly Leu 1 5 10 15 Val Lieu Ala Val Lieu Lleu Tyr Val Lys Arg Asn. Ser Ile Lys Glu Lieu 2O 25 30 Leu Met Ser Asp Asp Gly Asp Ile Thr Ala Val Ser Ser Gly Asn Arg 35 40 45 Asp Ile Ala Glin Val Val Thr Glu Asn. Asn Lys Asn Tyr Lieu Val Lieu 50 55 60 Tyr Ala Ser Glin Thr Gly. Thir Ala Glu Asp Tyr Ala Lys Llys Phe Ser 65 70 75 8O Lys Glu Lieu Val Ala Lys Phe Asn Lieu. Asn. Wal Met Cys Ala Asp Wal 85 90 95 Glu Asn Tyr Asp Phe Glu Ser Leu Asn Asp Val Pro Val Ile Val Ser 100 105 110 Ile Phe Ile Ser Thr Tyr Gly Glu Gly Asp Phe Pro Asp Gly Ala Val 115 120 125 Asn Phe Glu Asp Phe Ile Cys Asn Ala Glu Ala Gly Ala Leu Ser Asn 130 135 1 4 0 Leu Arg Tyr Asn Met Phe Gly Leu Gly Asn Ser Thr Tyr Glu Phe Phe 145 15 O 155 160 Asn Gly Ala Ala Lys Lys Ala Glu Lys His Leu Ser Ala Ala Gly Ala 1.65 170 175 Ile Arg Lieu Gly Lys Lieu Gly Glu Ala Asp Asp Gly Ala Gly Thir Thr 18O 185 19 O Asp Glu Asp Tyr Met Ala Trp Lys Asp Ser Ile Leu Glu Val Lieu Lys 195 200 2O5 Asp Glu Lieu. His Leu Asp Glu Glin Glu Ala Lys Phe Thr Ser Glin Phe 210 215 220 Gln Tyr Thr Val Leu Asn Glu Ile Thr Asp Ser Met Ser Leu Gly Glu 225 230 235 240 Pro Ser Ala His Tyr Lieu Pro Ser His Glin Lieu. Asn Arg Asn Ala Asp 245 250 255 Gly Ile Gln Leu Gly Pro Phe Asp Leu Ser Gln Pro Tyr Ile Ala Pro 260 265 27 O Ile Val Lys Ser Arg Glu Lieu Phe Ser Ser Asn Asp Arg Asn. Cys Ile 275 280 285 His Ser Glu Phe Asp Leu Ser Gly Ser Asn Ile Lys Tyr Ser Thr Gly 29 O 295 3OO Asp His Leu Ala Val Trp Pro Ser Asn Pro Leu Glu Lys Val Glu Glin 305 310 315 320 US 2005/0003473 A1 Jan. 6, 2005 71
-continued Phe Leu Ser Ile Phe Asn Lieu. Asp Pro Glu Thir Ile Phe Asp Leu Lys 325 330 335 Pro Leu Asp Pro Thr Val Lys Val Pro Phe Pro Thr Pro Thr Thr Ile 340 345 35 O Gly Ala Ala Ile Lys His Tyr Lieu Glu Ile Thr Gly Pro Val Ser Arg 355 360 365 Glin Leu Phe Ser Ser Lieu. Ile Glin Phe Ala Pro Asn Ala Asp Wall Lys 370 375 38O Glu Lys Lieu. Thir Lieu Lleu Ser Lys Asp Lys Asp Glin Phe Ala Val Glu 385 390 395 400 Ile Thr Ser Lys Tyr Phe Asn. Ile Ala Asp Ala Leu Lys Tyr Lieu Ser 405 410 415 Asp Gly Ala Lys Trp Asp Asn Val Pro Met Glin Phe Leu Val Glu Ser 420 425 43 O Val Pro Gln Met Thr Pro Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Leu 435 4 40 4 45 Ser Glu Lys Glin Thr Val His Val Thr Ser Ile Val Glu Asin Phe Pro 450 455 460 Asn Pro Glu Leu Pro Asp Ala Pro Pro Gly Val Gly Val Thr Thr Asn 465 470 475 480 Leu Lieu Arg Asn. Ile Glin Leu Ala Glin Asn. Asn. Wall Asn. Ile Ala Glu 485 490 495 Thr Asn Lieu Pro Wal His Tyr Asp Lieu. Asn Gly Pro Arg Lys Lieu Phe 5 OO 505 51O. Ala Asn Tyr Lys Lieu Pro Wal His Val Arg Arg Ser Asn. Phe Arg Lieu 515 52O 525 Pro Ser Asn Pro Ser Thr Pro Val Ile Met Ile Gly Pro Gly Thr Gly 530 535 540 Val Ala Pro Phe Arg Gly Phe Ile Arg Glu Arg Val Ala Phe Leu Glu 545 550 555 560 Ser Glin Lys Lys Gly Gly Asn. Asn. Wal Ser Lieu Gly Lys His Ile Lieu 565 570 575 Phe Tyr Gly Ser Arg Asn. Thir Asp Asp Phe Leu Tyr Glin Asp Glu Trp 58O 585 59 O Pro Glu Tyr Ala Lys Lys Lieu. Asp Gly Ser Phe Glu Met Val Val Ala 595 600 605 His Ser Arg Lieu Pro Asn. Thir Lys Llys Val Tyr Val Glin Asp Llys Lieu 610 615 62O Lys Asp Tyr Glu Asp Glin Val Phe Glu Met Ile Asn. Asn Gly Ala Phe 625 630 635 640 Ile Tyr Val Cys Gly Asp Ala Lys Gly Met Ala Lys Gly Val Ser Thr 645 650 655 Ala Lieu Val Gly Ile Leu Ser Arg Gly Lys Ser Ile Thir Thr Asp Glu 660 665 67 O Ala Thr Glu Lieu. Ile Lys Met Leu Lys Thr Ser Gly Arg Tyr Glin Glu 675 680 685 Asp Val Trp 69 O.
<210> SEQ ID NO 41 &2 11s LENGTH 693 &212> TYPE PRT US 2005/0003473 A1 Jan. 6, 2005 72
-continued <213> ORGANISM: Aspergillus niger P450 reductase <400 SEQUENCE: 41 Met Ala Glin Lieu. Asp Thr Lieu. Asp Lieu Val Val Lieu Ala Val Lieu Lieu 1 5 10 15 Val Gly Ser Val Ala Tyr Phe Thr Lys Gly Thr Tyr Trp Ala Val Ala 2O 25 30 Lys Thr Arg Met Pro Leu Pro Ala Pro Arg Met Asn Gly Ala Ala Lys 35 40 45 Ala Gly Lys Thr Arg Asn. Ile Ile Glu Lys Met Glu Glu Thr Gly Lys 50 55 60 Asn Cys Val Ile Phe Tyr Gly Ser Glin Thr Gly. Thr Ala Glu Asp Tyr 65 70 75 8O Ala Ser Arg Lieu Ala Lys Glu Gly Ser Glin Arg Phe Gly Lieu Lys Thr 85 90 95 Met Val Ala Asp Leu Glu Glu Tyr Asp Tyr Glu Asn Lieu. Asp Glin Phe 100 105 110 Pro Glu Asp Llys Val Ala Phe Phe Val Leu Ala Thr Tyr Gly Glu Gly 115 120 125 Glu Pro Thr Asp Asn Ala Val Glu Phe Tyr Glin Phe Phe Thr Gly Asp 130 135 1 4 0 Asp Wall Ala Phe Glu Ser Ala Ser Ala Asp Glu Lys Pro Leu Ser Lys 145 15 O 155 160 Leu Lys Tyr Val Ala Phe Gly Leu Gly Asn Asn Thr Tyr Glu His Tyr 1.65 170 175 Asn Ala Met Val Arg Glin Val Asp Ala Ala Phe Glin Lys Lieu Gly Pro 18O 185 19 O Glin Arg Ile Gly Ser Ala Gly Glu Gly Asp Asp Gly Ala Gly Thr Met 195 200 2O5 Glu Glu Asp Phe Leu Ala Trp Lys Glu Pro Met Trp Ala Ala Leu Ser 210 215 220 Glu Ser Met Asp Leu Glu Glu Arg Glu Ala Val Tyr Glu Pro Val Phe 225 230 235 240 Cys Val Thr Glu Asn Glu Ser Leu Ser Pro Glu Asp Glu Thr Val Tyr 245 250 255 Leu Gly Glu Pro Thr Glin Ser His Leu Glin Gly Thr Pro Lys Gly Pro 260 265 27 O Tyr Ser Ala His Asn Pro Phe Ile Ala Pro Ile Ala Glu Ser Arg Glu 275 280 285 Leu Phe Thr Val Lys Asp Arg Asn. Cys Lieu. His Met Glu Ile Ser Ile 29 O 295 3OO Ala Gly Ser Asn Lieu Ser Tyr Glin Thr Gly Asp His Ile Ala Val Trp 305 310 315 320 Pro Thr Asn Ala Gly Ala Glu Val Asp Arg Phe Leu Glin Val Phe Gly 325 330 335 Leu Glu Gly Lys Arg Asp Ser Val Ile Asn. Ile Lys Gly Ile Asp Wal 340 345 35 O Thr Ala Lys Val Pro Ile Pro Thr Pro Thr Thr Tyr Asp Ala Ala Val 355 360 365 Arg Tyr Tyr Met Glu Val Cys Ala Pro Val Ser Arg Glin Phe Val Ala 370 375 38O US 2005/0003473 A1 Jan. 6, 2005 73
-continued
Thr Telu Ala Ala Phe Ala Pro Met Arg Lys Ala Arg Glin Arg Lieu Cys 385 390 395 400
Wall Trp Wall Ala Glin Gly Teu Phe Pro Arg Glu Gly His Glin Pro Met 405 410 415
Teu Glin His Ala Glin Ala Teu Glin Ser Ile Thr Ser Pro Phe Ser 420 425 43 O
Ala Wall Pro Phe Ser Teu Teu Ile Glu Gly Ile Thr Lys Telu Glin Pro 435 4 40 4 45
Arg Tyr Tyr Ser Ile Ser Ser Ser Ser Telu Wall Glin Asp Ile 450 455 460
Ser Ile Thr Ala Wall Wall Glu Ser Wall Arg Teu Pro Gly Ala Ser His 465 470 475 480
Met Wall Gly Wall Thr Thr Asn Tyr Telu Teu Ala Teu Glin 485 490 495
Glin Asn Gly Arg Ser Teu Ser Pro Ser Arg Teu Asp Telu Telu His 5 OO 505
His Gly Pro Arg Asn Tyr Asp Gly Ile His Wall Pro Wall His Wall 515 525
Arg His Ser Asn Phe Teu Pro Ser Asp Pro Ser Arg Pro Ile Ile 530 535 540
Met Wall Gly Pro Gly Thr Gly Wall Ala Pro Phe Arg Gly Phe Ile Glin 545 550 555 560
Glu Arg Ala Ala Teu Ala Ala Lys Gly Glu Wall Gly Pro Thr Wall 565 570 575
Teu Phe Phe Gly Cys Arg Lys Ser Asp Glu Asp Phe Teu Tyr Asp 585 59 O
Glu Trp Lys Thr Tyr Glin Glin Telu Gly Asp Asn Teu Ile Ile 595 600 605
Thr Ala Phe Ser Glu Gly Pro Glin Lys Wall Tyr Wall Glin His Arg 610 615
Teu Arg Glu His Ser Glu Teu Wall Ser Asp Teu Teu Glin Ala 625 630 635 640
Thr Phe Tyr Wall Cys Gly Ala Ala Asn Met Ala Arg Glu Wall Asn 645 650 655
Teu Wall Telu Gly Glin Ile Ile Ala Ala Glin Arg Gly Teu Pro Ala Glu 660 665 67 O
Gly Glu Glu Met Wall Lys His Met Arg Arg Arg Gly Arg Glin 675 680 685
Glu Asp Wall Trp Ser 69 O.
SEQ ID NO 42 LENGTH 678 TYPE PRT ORGANISM: mus musculus
<400 SEQUENCE: 42
Met Gly Asp Ser His Glu Asp Thr Ser Ala Thr Wall Pro Glu Ala Wall 1 5 10 15
Ala Glu Glu Wal Ser Leu Phe Ser Thr Thr Asp Ile Wall Leu Phe Ser 25 30 Leu Ile Val Gly Val Leu Thir Tyr Trp Phe Ile Phe Lys Lys Lys Lys 35 40 45 US 2005/0003473 A1 Jan. 6, 2005 74
-continued
Glu Glu Ile Pro Glu Phe Ser Lys Ile Glin Thr Thr Ala Pro Pro Val 50 55 60 Lys Glu Ser Ser Phe Val Glu Lys Met Lys Lys Thr Gly Arg Asn. Ile 65 70 75 8O Ile Val Phe Tyr Gly Ser Gln Thr Gly Thr Ala Glu Glu Phe Ala Asn 85 90 95 Arg Lieu Ser Lys Asp Ala His Arg Tyr Gly Met Arg Gly Met Ser Ala 100 105 110 Asp Pro Glu Glu Tyr Asp Leu Ala Asp Leu Ser Ser Leu Pro Glu Ile 115 120 125 Asp Llys Ser Lieu Val Val Phe Cys Met Ala Thr Tyr Gly Glu Gly Asp 130 135 1 4 0 Pro Thr Asp Asn Ala Glin Asp Phe Tyr Asp Trp Leu Glin Glu Thir Asp 145 15 O 155 160 Val Asp Lieu. Thr Gly Wall Lys Phe Ala Val Phe Gly Lieu Gly Asn Lys 1.65 170 175 Thr Tyr Glu His Phe Asn Ala Met Gly Lys Tyr Val Asp Glin Arg Leu 18O 185 19 O Glu Gln Leu Gly Ala Glin Arg Ile Phe Glu Lieu Gly Lieu Gly Asp Asp 195 200 2O5 Asp Gly Asn Lieu Glu Glu Asp Phe Ile Thir Trp Arg Glu Glin Phe Trp 210 215 220 Pro Ala Val Cys Glu Phe Phe Gly Val Glu Ala Thr Gly Glu Glu Ser 225 230 235 240 Ser Ile Arg Glin Tyr Glu Leu Val Val His Glu Asp Met Asp Thr Ala 245 250 255 Lys Val Tyr Thr Gly Glu Met Gly Arg Lieu Lys Ser Tyr Glu Asn Glin 260 265 27 O Lys Pro Pro Phe Asp Ala Lys Asn Pro Phe Leu Ala Ala Val Thr Thr 275 280 285 Asn Arg Lys Lieu. Asn Glin Gly Thr Glu Arg His Leu Met His Leu Glu 29 O 295 3OO Leu Asp Ile Ser Asp Ser Lys Ile Arg Tyr Glu Ser Gly Asp His Val 305 310 315 320 Ala Val Tyr Pro Ala Asn Asp Ser Thr Lieu Val Asn Glin Ile Gly Glu 325 330 335 Ile Leu Gly Ala Asp Leu Asp Wal Ile Met Ser Lieu. Asn. Asn Lieu. Asp 340 345 35 O Glu Glu Ser Asn Lys Lys His Pro Phe Pro Cys Pro Thr Thr Tyr Arg 355 360 365 Thr Ala Leu Thr Tyr Tyr Leu Asp Ile Thr Asn Pro Pro Arg Thr Asn 370 375 38O Val Leu Tyr Glu Leu Ala Glin Tyr Ala Ser Glu Pro Ser Glu Glin Glu 385 390 395 400 His Lieu. His Lys Met Ala Ser Ser Ser Gly Glu Gly Lys Glu Lieu. Tyr 405 410 415 Leu Ser Trp Val Val Glu Ala Arg Arg His Ile Leu Ala Ile Leu Glin 420 425 43 O Asp Tyr Pro Ser Leu Arg Pro Pro Ile Asp His Lieu. Cys Glu Lieu Lieu 435 4 40 4 45