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(12) Patent Application Publication (10) Pub. No.: US 2008/0064084 A1 MULLER Et Al US 20080064084A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0064084 A1 MULLER et al. (43) Pub. Date: Mar. 13, 2008 (54) METHOD FOR PRODUCING Related U.S. Application Data RECOMBINANT TRYPSIN (62) Division of application No. 10/470,508, filed on Apr. 12, 2004, now Pat. No. 7,276,605. (75) Inventors: Rainer MULLER, Penzberg (DE): Stephan GLASER, Seeshaupt (DE); (30) Foreign Application Priority Data Frank GEIPEL, Penzberg (DE); Johann-Peter THALHOFER, Feb. 1, 2002 (EP)............................. PCT/EPO2/O1072 Weilheim (DE); Bernhard REXER, Feb. 1, 2001 (EP)........................................ O1102342.1 Weilhem (DE); Claus SCHNEIDER, Eppelheim (DE); Michael RATKA, Publication Classification Mannheim (DE); Stephanie RONNING, Penzberg (DE); Hellmut (51) Int. Cl. ECKSTEIN, Weilheim (DE); Claudia CI2N I/19 (2006.01) GIESSEL, Greiling (DE) C7H 2L/00 (2006.01) C07K I4/00 (2006.01) CI2N I/00 (2006.01) Correspondence Address: (52) U.S. Cl. .................... 435/254.21: 435/243: 530/350; BARNES & THORNBURG LLP (Roche) 536/23.5 11 SOUTH MERIDAN STREET INDIANAPOLIS, IN 46204 (US) (57) ABSTRACT The present invention concerns a method for producing (73) Assignee: ROCHE DIAGNOSTICS OPERA recombinant trypsin from porcine pancreas in Pichia pas toris which is soluble and secreted into the culture medium, TIONS, INC., Indianapolis, IN (US) whereby expression at pH 3.0-4.0 substantially prevents (21) Appl. No.: 11/853,483 activation of trypsinogen to B-trypsin and autolysis of B-trypsin by C-trypsin into e-trypsin and from there into (22) Filed: Sep. 11, 2007 inactive peptides. 820 C Bat Patent Application Publication Mar. 13, 2008 Sheet 1 of 3 US 2008/0064084 A1 Fig. 1 4280 C. Bg s sa Rica. si Sfc. Wales 4000 EC 36 C Bar W Sac Pla 2-ISS Bg CII E.0.si Bs A cert Nsp ca SspB C thacIN HindIII beeRW ? s Stu s Saat d Da Sae c t Bgll s XhoI se E. 2coR NaeAcy a Baar ea Sex al OOO Sg ssp. So SO na Acy N Styl f car bac an Hale C 2000 C. Say r Bing Pia Ea Patent Application Publication Mar. 13, 2008 Sheet 2 of 3 US 2008/0064084 A1 Figur 2 Patent Application Publication Mar. 13, 2008 Sheet 3 of 3 US 2008/0064084 A1 Figur 3 US 2008/0064084 A1 Mar. 13, 2008 METHOD FOR PRODUCING RECOMBINANT component of some pharmaceutical preparations (ointments, TRYPSIN dragees and aerosols for inhalation (“Rote Liste', 1997: The United States Pharmacopeia, The National Formulary, CROSS-REFERENCE TO RELATED USP23-NF18, 1995)). Since the use of enzymes from animal APPLICATION Sources is no longer permitted in many cases (potential contamination with infectious agents), recombinant trypsin 0001. This application is a divisional of U.S. patent molecules for the desired biotechnological applications have application Ser. No. 10/470,508, filed Apr. 12, 2004, which is a U.S. national counterpart application of international to be provided from microbial hosts. application Serial No. PCT/EP02/01072 filed Feb. 1, 2002, 0006 There are several methods for the recombinant which claims the benefit of European application no. production of trypsin from various organisms. 01102342.1 filed Feb. 1, 2001. 0007 Graf, L. et al (1987 and 1988) describe the expres 0002 The invention concerns a method for the recombi sion and secretion of rat trypsin and trypsinogen mutants in nant production of trypsin. For this purpose a trypsinogen E. coli. In order to secrete the trypsinogen molecules into the with a shortened propeptide sequence is preferably periplasm of E. coli the native trypsinogen signal sequence expressed in a recombinant host cell and secreted into the is replaced by the signal sequence of the bacterial alkaline culture medium in an uncleaved form. Subsequently the phosphatase (phoA). The Secreted inactive trypsinogen mol propeptide sequence is cleaved in a controlled manner to ecules are isolated from the periplasm and activated by form active trypsin. enzymatic cleavage using purified enterokinase. 0003) Trypsin is a serine protease which catalyses a 0008 Vasquez, J. R. et al. (1989) describe the expression hydrolytic cleavage of peptides at the carboxyl group of the and secretion of anionic rat trypsin and trypsin mutants in E. basic amino acids arginine and lysine (Keil B., 1971). coli. In order to express and secrete the active trypsin Trypsin from bovine pancreas was one of the first proteolytic molecules into the periplasm of E. coli, the native trypsi enzymes that could be used in a pure form and in adequate nogen prepro segment (signal sequence and activation pep quantities for exact chemical and enzymatic studies tide) is replaced by the signal sequence of the bacterial (Northrop et al., 1948). It was subsequently also possible to alkaline phosphatase (phoA) and the phoA promoter that can isolate proteases that can be allocated to the trypsin family be regulated by phosphate is used. Active trypsin is isolated from other higher vertebrates (pig Charles et al. 1963/ from the periplasm. However, the yield is very low (ca. 1 sheep Bricteux-Gregoire et al. (1966); Travis (1968)/tur mg/l). key —Ryan (1965)/humans Travis et al. (1969) and oth ers). At this time the first enzymes belonging to the trypsin 0009 Higaki, J. N. et al. (1989) describe the expression family were also isolated from two species of Streptomyces and secretion of trypsin and trypsin mutants into the peri (Morihara and Tsuzuki (1968); Trop and Birk (1968); plasm of E. coli using the tac promoter and the Styphimu Wählby and Engström (1968); Wählby (1968; Jurasek et al. rium his signal sequence. The yield of active trypsin is ca. (1969)). 0.3 mg/l. The volume yield of active anionic rat trypsin can be increased to about 50 mg/1 by high cell density fermen 0004 The enzyme is synthesized in the pancreas cells of tation (Yee, L. and Blanch, H. W., (1993)). However, the vertebrates as an inactive precursor, trypsinogen, and Sub authors refer to problems in the expression and secretion of sequently converted into the active form by cleavage of the active trypsin in E. coli. Enzymatically active trypsin is propeptide (Northrop et al. (1948), Desnuelle (1959)). The formed in the periplasm of E. coli after cleavage of the signal first trypsinogen molecules were activated naturally by the sequence and native trypsin protein folding to form 6 enteropeptidase enterokinase which hydrolyses the peptide disulfide bridges. The formation of active trypsin is toxic for bond between (Asp)-Lys--Ile which cleaves off the the cell since active trypsin hydrolyses the periplasmatic E. propeptide (Keil (1971)). The recognition sequence of the coli proteins which lyses the cells. Moreover the protein enterokinase (Asp)-Lys is accordingly located directly at folding of trypsin and in particular the correct native for the C-terminus of the propeptide in almost all previously mation of the 6 disulfide bridges appears to be impeded in known trypsinogen molecules (Light et al. (1980)). The the periplasm of E. coli. The system is not suitable for the activation process can also proceed autocatalytically at isolation of relatively large amounts of trypsin (>10 mg: physiological pH values since a lysine is located on the Willett, W. S. et al., (1995)). C-terminal side of the enterokinase recognition sequence and hence the Lys--Ile peptide bond can also be hydrolyzed 0010. In order to produce larger amounts of trypsin by trypsin (Light et al. (1980)). (50-100 mg) for X-ray crystallographic investigations, an 0005 Trypsin has always been an interesting protease for inactive trypsinogen precursor is produced in yeast under the biotechnological applications due to its ready availability control of a regulatable ADH/GAPDH promoter and from various mammals, high specificity (only cleaves at the secreted by fusion with the yeast C. factor leader sequence. C-terminal side of lysine or arginine) together with high The expression product secreted into the medium is con specific activity (~150 U/mg) and its good storage stability. verted quantitatively into trypsin in vitro by means of Trypsin is mainly used for the tryptic cleavage of peptides enterokinase. The yield is 10-15 mg/l (Hedstrom, L. et al. into Small sections for sequencing, for detaching adherent (1992)). cells from coated cell culture dishes and for cleaving fusion 0011 DNA sequences are described in EP 0 597 681 proteins into the target peptide and the fusion component, which code for mature bovine trypsin and bovine trypsino for activating propeptides (e.g. trypsinogen to trypsin) and gen with an initial methionine residue. In addition the for the recombinant production of peptide hormones (e.g. expression in E. coli is described but the strategy of how proinsulin to insulin, cf. WO 99/10503). Trypsin is also a active trypsin is formed in E. coli is not explained. US 2008/0064084 A1 Mar. 13, 2008 0012. A method for producing trypsin from porcine pan 0017. The object according to the invention is achieved creas or a derivative thereof in Aspergillus by a recombinant by a method for the recombinant production of trypsin method is described in WO 97/00316. A vector is used for comprising the steps: transformation which codes for trypsinogen or a derivative thereof which is fused at the N-terminus to a functional 0018 a) transforming a host cell with a recombinant signal peptide. However, yeast cultures achieve higher bio nucleic acid which codes for a trypsinogen with an enter mass concentrations compared to Aspergillus cultures and okinase recognition site in the propeptide sequence in a grow considerably more rapidly and thus the specific expres secretable form, preferably fused with a signal peptide that sion yield per yeast cell can be less than that for Aspergillus mediates secretion, cells in order to achieve yields that are required for an 0019 b) culturing the host cell under conditions which economic expression method.
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