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 (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 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.

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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 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 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 and (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 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 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 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 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. enable an expression of the recombinant nucleic acid and a 0013 A method for the recombinant production of a secretion of the expression product into the culture medium, Zymogenic precursor of proteases which contains an auto the conditions being selected Such that an autocatalytic catalytic cleavage site that does not occur naturally is cleavage of the propeptide sequence is at least Substantially described in WO 99/10503. The method comprises an prevented, expression of the Zymogenic precursor in E. coli in the form of inclusion bodies with Subsequent purification and rena 0020 c) isolating the expression product from the culture turation under conditions where the protease part of the medium, Zymogenic precursor is formed in its natural conformation 0021 d) cleaving the propeptide sequence to form active and the cleavage of the renatured Zymogenic precursor then trypsin and occurs autocatalytically. A disadvantage of this method are the large losses that often occur during renaturation and the 0022 e) optionally separating non-cleaved trypsinogen large Volumes that are required for an industrial production. molecules from active trypsin. 0014. The recombinant production of trypsin analogues 0023 The host cell used in the method according to the in Pichia pastoris is described in WO 00/17332. A vector is invention can be a eukaryotic or prokaryotic host cell like used for the transformation which codes for a trypsinogen those known from the prior art. The host cell is preferably a analogue (derivative of bovine trypsinogen) in which the eukaryotic cell, particularly preferably a fungal cell and lysine at the C-terminus of the propeptide was most preferably a yeast cell. Suitable examples of yeast cells exchanged by mutation for another amino acid (apart from are Pichia pastoris, Hansenula polymorpha, Saccharomyces arginine or lysine) and which is fused N-terminally to a cerevisiae, Schizosaccharomyces pombae where methy functional signal peptide. In this method the trypsin ana lotrophic yeasts such as Pichia pastoris or Hansenula poly logues are secreted into the medium in a soluble form and as morpha and in particular Pichia pastoris are preferably a result of the incorporated mutation are also not activated used. and further degraded by undesired autocatalysis even at relatively high pH values of the fermentation process. An 0024. It is expedient to use a host cell that is able to aminopeptidase is then used for activation. However, a express the recombinant nucleic acid and to secrete the disadvantage of this method is the need to remove the expression product into the culture medium under conditions additional aminopeptidase which may have disadvantageous where an autocatalytic cleavage of the propeptide sequence side activities for the subsequent use of the trypsin in the is at least Substantially and preferably essentially quantita final process. tively prevented. Such conditions advantageously comprise an acidic pH value of the culture medium, particularly 0.015 The expression of naturally occurring trypsinogen preferably a pH value in the range between 3 and 4. genes in Pichia pastoris is described in WO 01/55429. In Trypsinogen is stable at acidic pH values and especially at order to avoid autocatalytic activation the fermentation is pH values of up to 4 whereas trypsin is autocatalytically carried out in the example in the less acid pH range at ca. 6. activated in a neutral or alkaline medium (Keil, B. (1971)). It is thus outside the optimal pH range which prevents autocatalytic activation during longer run times in the 0025 The culture under suitable conditions that occurs in expression phase. Other disadvantages of the method are the the method according to the invention prevents a premature trypsinogen genes that are not codon-optimized for expres activation of the recombinant trypsinogen secreted by the sion in Pichia pastoris and the associated low expression host cell which can also substantially prevent further deg yields. radation of the resulting trypsin to as far as inactive peptides. The stability of the recombinant trypsinogen under the 0016. The object of the invention is to provide a method culture conditions is important since the expression should for the recombinant production of trypsin in which the be accompanied by a secretion. In this connection secretion disadvantages of the prior art are at least partially eliminated from the host cell is understood as the discharge of trypsi and which allows active trypsin to be obtained in a simple nogen from the cytoplasm through the cell membrane into manner in a high yield and activity. In particular it should be the culture medium. This usually occurs by N-terminal possible to isolate trypsinogen as an intermediate product in fusion of the trypsinogen with a functional signal peptide. a soluble form from the culture medium of the expression Examples of Suitable signal peptides are known signal host which should not be subject to any substantial degree of peptides from yeast and especially preferably the signal autocatalytic activation during the fermentation and purifi peptide of the a factor from Saccharomyces cerevisiae. cation. Furthermore it should be possible to subsequently However, it is of course also possible to use other signal activate the trypsinogen autocatalytically and/or by adding peptides e.g. the signal peptide which naturally controls the recombinant trypsin. secretion of trypsinogen. US 2008/0064084 A1 Mar. 13, 2008

0026. In order to improve the expression yield of the range of 3-4 the trypsinogen formed by expression of the recombinant trypsin, a recombinant nucleic acid with an recombinant nucleic acid accumulates in an intact form in optimized codon usage for the respective host cell is pref the culture medium. erably used. Accordingly it is advantageous to use a nucleic 0031. The culture conditions in the method according to acid for a yeast cell which has an optimized codon usage for the invention are selected Such that autocatalytic cleavage of yeast. Such nucleic acids having an optimized codon usage the propeptide sequence is substantially prevented. After can for example be produced by synthesizing individual isolating the expression product from the culture medium, oligonucleotides in parallel and Subsequently combining the propeptide sequence can be cleaved off under controlled them. conditions. This cleavage can for example be carried out by adding recombinant trypsin or/and by autocatalytic cleav 0027. The method according to the invention is basically age. In this connection autocatalytic cleavage is understood Suitable for producing any types of recombinant trypsin as the self-activation of recombinant trypsinogen which may provided the respective trypsinogen that is secreted as an be optionally accelerated by adding Small amounts of expression product by the host cell is essentially stable under recombinant trypsin but without adding a foreign protein. In the culture conditions. The method is preferably used to this manner it is not necessary to use an additional foreign produce trypsin from vertebrates, in particular from mam protein which is usually derived from animal raw material mals such as pigs, sheep or humans. Porcine trypsin is Sources and has to be Subsequently removed again which particularly preferably produced. could cause undesired cleavages in the Subsequent applica tion. The autocatalytic activation preferably occurs at a pH 0028. Furthermore it is preferred that a recombinant in the range of 7-8. This ensures that the trypsinogen formed nucleic acid is used which codes for a trypsinogen with a during the expression can be firstly highly purified in a shortened propeptide sequence. Trypsinogen is understood strongly acidic range and the activation can be specifically as a protein which, although it has formed a Substantially started by rebuffering preferably in the presence of small correct protein structure, has no or only a very low pro amounts e.g. 20 mM CaCl to a neutral to weakly basic pH teolytic activity compared to active trypsin which is advan range. The activation can be stopped by changing the pH tageously at least 5-fold and particularly preferably at least again to a strongly acidic range. Trypsin can for example be 10-fold less than the proteolytic activity of the active form. purified by chromatography on ion exchanger material Such The natural length of the propeptide part, e.g. 25 amino acids as SP-Sepharose RXL or benzamidine-Sepharose. in the case of a porcine trypsinogen, is shortened preferably down to a propeptide sequence which only consists of a 0032. In order to purify the expression product from the recognition sequence for an enteropeptidase e.g. enteroki culture medium, the culture Supernatant can be firstly sepa nase e.g. an amino acid sequence Asp-Asp-Asp-Asp-Lys. A rated from the cells as described in example 6.1. The few additional amino acids that are due to the cloning, e.g. Subsequent purification comprising an autocatalytic activa up to 5 amino acids, can be optionally attached to the tion of the trypsinogen is preferably carried out by suitable N-terminus of the enterokinase recognition sequence. chromatographic purification procedures. In a particularly preferred embodiment a chromatography is carried out as 0029. It was found that the expression of a shortened described in example 6.2. without prior separation of the recombinant trypsinogen according to the invention contain cells in which a buffer containing calcium ions preferably at ing the amino acids Glu-Phe attached due to the cloning to a final concentration of 1-30 mM calcium is added to the the N-terminus of the enterokinase recognition site results in culture medium which still contains the cells. Subsequently significantly higher yields than is the case for the expression steps (c), (d) and optionally (e) of the method according to of natural trypsinogen in the method according to the the invention are carried out for example using Suitable invention. Hence in a particularly preferred embodiment of chromatographic purification procedures and a rebuffering the method according to the invention a recombinant nucleic step for the autocatalytic cleavage of the expression product. acid having the nucleotide sequence shown in SEQ ID NO.22 is used which codes for a porcine trypsinogen with 0033 Hence a particularly preferred embodiment of the a shortened propeptide sequence. method according to the invention is characterized by: 0030. In the method according to the invention the 0034) a) transforming a host cell with a recombinant expression of the recombinant trypsinogen is preferably nucleic acid which codes for the Zymogenic precursor controlled by an inducible expression control sequence. trypsinogen from the pig which is fused with a signal peptide Hence the growth of the host cell can take place before sequence and contains a propeptide which is shortened down induction of expression under conditions which are favour to the enterokinase recognition sequence, where this cleav able or optimal for the growth of the host cell. Thus for age site is cleaved by recombinant trypsin or is autocata example growth can take place at pH 5-7, in particular pH lytically cleaved under certain buffer conditions and the 5-6 up to a predetermined optical density during which the shortened trypsinogen can be cleaved to form active trypsin regulatable expression control sequence is repressed. in this process, Expression can then be induced by changing the temperature 0035) b) culturing the host cell during the expression and/or by adding an inducer. An example of a preferred phase at an acidic pH, preferably pH 3-4 so that the expression control sequence is the AOX 1- promoter from shortened trypsinogen is present in a soluble form and Pichia pastoris that can be induced by methanol which is secreted into the culture medium but the autocatalytic acti particularly suitable for inducible expression in methy Vation is substantially prevented, lotrophic yeasts. The culture conditions are advantageously changed before induction of the expression control sequence 0036 c) isolating the shortened recombinant trypsinogen in Such a manner that for example by changing the pH to a from the culture Supernatant and activating it under condi US 2008/0064084 A1 Mar. 13, 2008

tions which allow an effective cleavage of the shortened the invention. The recombinant trypsinogen has a propeptide trypsinogen by recombinant trypsin or by autocatalytic sequence which is shortened compared to the natural cleavage and propeptide sequence and contains an enterokinase recogni tion site. The recombinant trypsinogen according to the 0037 d) optionally separating non-cleaved trypsinogen invention preferably has the amino acid sequence shown in molecules from active trypsin. SEQ ID NO. 23. 0038. The shortening of the propeptide described in this invention has a positive effect especially on expression and 0046) The present invention is also elucidated by the autocatalytic activation. Furthermore after the autocatalytic following figures and examples. activation, the undesired further autolysis increases signifi 0047 FIG. 1: shows a plasmid map of the expression cantly at a pH of >4.0 during the growth phase and expres plasmid pTRYP-9 containing the complete recombinant sion phase of the culture. trypsinogen, 0039. An additional increase in the expression yield in 0048 FIG. 2: shows a plasmid map of the expression the method according to the invention can be achieved by plasmid pTRYP-11 containing the shortened recombinant transforming the host cell with several vectors each of which trypsinogen (sh-trypsinogen) and the Zeocin resistance contains a recombinant nucleic acid as stated above where marker gene (ZeoR), the vectors contain different selection markers e.g. Zeocin 0049 FIG. 3: shows a plasmid map of the expression and G418. Culture under multiple selection conditions sur plasmid pTRYP-13 containing the shortened recombinant prisingly allows the expression yield of recombinant trypsi trypsinogen (sh-trypsinogen) and a kanamycin/G418 selec nogen to be increased considerably further. tion marker gene (KanR). 0040 Another subject matter of the invention is a recom binant nucleic acid which codes for a trypsinogen having an EXAMPLES enterokinase recognition site in the propeptide sequence where the propeptide sequence is shortened relative to the Methods: natural propeptide sequence and is preferably fused with a Recombinant DNA Techniques signal peptide sequence. The shortened propeptide sequence according to the invention preferably consists of an enter 0050 Standard methods were used to manipulate DNA as okinase recognition site having the amino acid sequence described by Sambrook, J. et al. (1989) in Molecular Clon Asp-Asp-Asp-Asp-Lys and optionally up to 5 additional ing: A Laboratory Manual. Cold Spring Harbor Laboratory amino acids located arranged N-terminally thereof. The Press, Cold Spring Harbor, N.Y. The molecular biological nucleic acid according to the invention particularly prefer reagents were used according to the manufacturers instruc ably has the nucleotide sequence shown in SEQ ID NO.22. tions. 0041. The nucleic acid according to the invention is Protein Determination preferably in operative linkage with a regulatable expression 0051. The protein determination of purified trypsin was control sequence which is for example a suitable expression carried out by measuring the absorbance at 280 nm. A value control sequence for gene expression in yeast cells Such as of 13.6 was used for A 1%/280 nm for 1 cm path length. the AOX1 promoter from Pichia pastoris. Calculation: 0042. The invention also concerns a recombinant vector which contains at least one copy of a recombinant nucleic Protein mg/ml=(10 mg/ml AA sample *dilution), 13.6 acid as stated above. The vector is preferably a vector that Pichia pastoris and Expression Vectors for Pichia pastoris is suitable for gene expression in yeast cells. Examples of 0052 The catalogue and instruction manuals from Invit such vectors are described in Sambrook et al., Molecular rogen were used as instructions for handling Pichia pastoris cloning: A Laboratory Manual, Cold Spring Harbor Labo and the expression vectors. The vectors for expressing the ratory Press, Cold Spring Harbor, N.Y. shortened recombinant trypsinogen are based on the vectors 0043. In addition to the recombinant nucleic acid, the pPICZOA and pPIC9K from Invitrogen. vector contains other suitable genetic elements for the respective intended use and in particular a selection marker Example 1 gene. Vectors are particularly preferably used which can be present in the cell in multiple copies especially vectors 0053 Gene synthesis of the complete recombinant trypsi which can be integrated in a multiple form into the genome nogen with optimized codon usage for expression in yeast of the host cell. The invention also concerns combinations of 0054) One of the preferred methods for providing the vectors which each contain different selection marker genes method according to the invention is to synthesize a codon and which can be propagated concurrently in a host cell. optimized gene sequence. In order to optimize each codon for expression in yeast it was necessary to carry out a 0044) In addition the invention concerns a recombinant complete de novo synthesis of the ca. 700 bp gene which cell which is transformed with a nucleic acid according to codes for the complete recombinant trypsinogen. It was the invention or a vector according to the invention. The possible to optimize each codon when necessary by utilizing recombinant cell is preferably a yeast cell in particular a the degenerate code in the retranslation of the amino acid methylotrophic yeast cell Such as Pichia pastoris or sequence of porcine trypsinogen according to SEQID NO.1. Hansenula polymorpha. For this purpose the gene was divided into 18 oligonucle 0045 Finally the invention concerns a recombinant otides having a length of 54 to 90 nucleotides. The oligo trypsinogen which is coded by a nucleic acid according to nucleotides were designed as an alternating sequence of US 2008/0064084 A1 Mar. 13, 2008

sense strand and counter-strand fragments whose 5' and 3' Example 3 ends each overlapped in a complementary manner with the Cloning the PCR Fragments of the Complete Recombinant neighboring oligonucleotides. The overlapping region was Trypsinogen Generated by Gene Synthesis and of the Short selected in each case such that unspecific binding was ened Recombinant Trypsinogen into Expression Vectors for largely prevented in the annealing reaction in the Subsequent Pichia pastoris PCR reaction. The oligonucleotides at the 5' and 3' end of the 0.058. The PCR fragments were recleaved with EcoRI gene were provided upstream and downstream of the coding and Xbal (Roche Diagnostics GmbH), isolated again region with recognition sites for restriction endonucleases (QIAquick Gel Extraction Kit/Qiagen) and Subsequently which could be used for the later insertion of the synthetic ligated into a fragment of the expression vector pPICZOA gene according to SEQ ID NO.2 into expression vectors. (Invitrogen) linearized with EcoRI and Xbal (Roche Diag Thus a recognition site for the restriction endonuclease nostics GmbH) and isolated with the QIAquick Gel Extrac EcoRI was incorporated upstream and a recognition site for tion Kit/Qiagen. For this 1 ul (20 ng) vector fragment and 3 the restriction endonuclease Xbal was incorporated down ul (100 ng) PCR fragment, 1 Jul 10x buffer (Sambrook stream. The sequences of the oligonucleotides are shown in et al., 1989 B.27), 1 ul T4 DNA ligase, 4 ul sterile SEQ ID NO.3 to 20. H.O.sistined were pipetted, carefully mixed and incubated overnight at 16°C. In this vector the synthetic gene is under 0.055 Gene synthesis was carried out by means of the the control of the AOX1 promoter (promoter for the alcohol PCR reaction. For this the coding region was firstly divided oxidase 1 from Pichia pastoris) that can be induced with into three segments (oligonucleotides 3 to 8, 9 to 14, 15 to methanol (Mallinckrodt Baker B. V.) and is located in the 20) and these segments were generated in separate PCR correct reading frame behind the signal peptide of the a reactions using overlapping complementary oligonucle factor from Saccharomyces cerevisiae. In order to check this otides. In this process the gene fragment was extended in a and isolate the plasmid, 5 Jul of the ligation mixture were stepwise manner till the full length product was formed then transformed in 200 ul competent cells (Hanahan (1983) which was then amplified in Subsequent cycles. The anneal of E. coli XL1Blue (Stratagene). A 30 min incubation on ice ing temperature was selected according to the overlapping was followed by a heat shock (90 sec at 42° C.). Subse region with the lowest melting temperature. quently the cells were transferred to 1 ml LB medium and 0056. The three segments were subsequently analysed by incubated for 1 hour at 37°C. in LB medium for phenotypic agarose gel electrophoresis, the products having the expression. Aliquots of this transformation mixture were expected length were isolated from the gel by means of the plated out on LB plates using 100 g/ml Zeocin as the QIAquick Gel Extraction Kit (Qiagen) and synthesized to selection marker and incubated for 15 hours at 37° C. The form the complete gene product in a further PCR reaction. plasmids were isolated from the grown clones (Sambrook, J. The first 5 cycles of the PCR reaction were carried out et al. (1989) In. Molecular cloning: A Laboratory Manual. without adding the primer at the 5' end and at the 3' end of Cold Spring Harbor Laboratory Press, Cold Spring Harbor, the entire gene so that at first a few fragments of the gene N.Y.) and then checked for an error-free base sequence by product of the expected length were formed from the three means of restriction analysis and sequencing. The expres segments. The annealing temperature was selected accord sion vectors formed in this manner which contain a synthetic ing to the overlapping region with the lowest melting gene for the complete recombinant trypsinogen or a short temperature. Then the terminal primers were added and the ened recombinant trypsinogen were named pTRYP-9 (see annealing temperature was increased according to the FIG. 1) and pTRYP-11 (see FIG. 2). annealing temperature of the primer with the lowest melting Transformation of pTRYP-9 and pTRYP-11 into Pichia temperature. The gene fragment of the expected length was pastoris then amplified in a further 25 cycles. The PCR fragment was 0059 For the transformation of pTRYP-9 and pTRYP-11 checked by sequencing. into Pichia pastoris X-33, GS 115 or KM71H with subse quent integration into the genome, the vector was firstly Example 2 linearized with SacI (Roche Diagnostics GmbH). The trans formation was carried out by electroporation using the Gene Generation of the Shortened Trypsinogen Gene Pulser II (Biorad). For this a colony of Pichia pastoris was 0057 The codons for the first 20 amino acids of the inoculated with 5 ml YPD medium (Invitrogen) and incu naturally occurring trypsinogen were deleted by means of a bated overnight at 30° C. while shaking. The overnight specially designed 5' primer according to SEQ ID NO. 21 culture was subsequently reinoculated 1:2000 in 200 ml Such that only the codons for the recognition sequence of the fresh YPD medium (Invitrogen) and incubated overnight at enteropeptidase enterokinase Asp-Asp-Asp-Asp-Lys and the 30° C. while shaking until an ODoo of 1.3 to 1.5 was codons Glu-Phe necessary for cloning into the expression reached. The cells were centrifuged (1500xg/5 minutes) and vectors for Pichia pastoris due to the EcoRI restriction the pellet was resuspended in 200 ml ice-cold, sterile water endonuclease recognition sequence remain as the sequence (0° C.). The cells were again centrifuged (1500xg/5 min of the propeptide at the N-terminus of the shortened recom utes) and resuspended in 100 ml ice-cold, sterile water (0° binant trypsinogen. The deletion was introduced by a PCR C.). The cells were again centrifuged and resuspended in 10 reaction on the PCR fragment of the gene for the complete ml ice-cold (0°C.) 1 M sorbitol (ICN). The cells were again recombinant trypsinogen using the 5' primer according to centrifuged and resuspended in 0.5 ml ice-cold (0°C.) 1 M SEQ ID NO. 21 and the 3' primer according to SEQID NO. sorbitol (ICN). The cells isolated in this manner were kept 20. The DNA sequence and the amino acid sequence of the on ice and immediately used for the transformation. shortened recombinant trypsinogen are shown in SEQ ID 0060 80 ul of the cells were admixed with ca. 1 lug NO. 22 and SEQ ID NO. 23 respectively. linearized pTRYP-9 or pTRYP-11 vector DNA and the US 2008/0064084 A1 Mar. 13, 2008

entire mixture was transferred into an ice-cold (0° C.) gen. The migration properties of the new protein bands in the electroporation cuvette and incubated for a further 5 minutes expression clones corresponds to the calculated molecular on ice. Subsequently the cuvette was placed in the Gene weight and is slightly higher than that of bovine trypsin that Pulser II (Biorad) and transformation was carried out at 1 had been applied as the control marker. This slight difference kV, 1 kS2 and 25 uF. After electroporation the mixture was in size indicates an intact, non-activated trypsinogen. admixed with 1 ml 1 M sorbitol (ICN) and subsequently 100 to 150 ul was plated out on a YPDS agar plate (Invitrogen) 0067 Surprisingly the recombinant shortened trypsino containing 100 g/ml Zeocin (Invitrogen). The plates were gen was expressed significantly more strongly than the subsequently incubated for 2-4 days at 30° C. recombinant complete trypsinogen. 0061 The clones were reinoculated on raster MD (=mini Example 4 mal dextrose) plates and analysed further. Grown clones were picked, resuspended in 20 ul sterile water, lysed (1 Increasing the Expression Yield by Multiple Transformation hour, 30° C., 10 min, -80° C.) with 17.5 Ulyticase (Roche 0068 The best clones from the expression experiments Diagnostics GmbH) and directly examined for correct inte with the recombinant shortened trypsinogen were again gration of the synthetic trypsinogen expression cassette by prepared for electroporation as described above and again means of PCR. transformed with 1 uglinearized pTRYP-11 vector DNA and the transformation mixture was plated out on YPDS agar 0062) Clones which had integrated the complete expres plates (Invitrogen) containing 1000 to 2000 ug/ml Zeocin sion cassette during transformation into the genome, were (Invitrogen). This increases the selection pressure in Such a then used in expression experiments. way that only clones can grow that have integrated several Expression of the Complete and Shortened Recombinant copies of the expression vector pTRYP-11 into the genome Trypsinogen and hence also several copies of the respective resistance gene (in this case Zeocin R). The Zeocin R resistance protein 0063 Positive clones were inoculated in 3 ml BMGY is the product of the bleomycin gene from Streptoalloteichus medium (Invitrogen) and incubated overnight at 30° C. hindustanus (Calmels, T. et al., (1991); Drocourt, D. et al., while shaking. Subsequently the OD was determined at 600 (1990)), which binds Zeocin R in a stoichiometric concen nm and they were reinoculated in 10 ml BMMY medium tration ratio and hence makes the cell resistant to Zeocin R. (Invitrogen) pH 3.0 such that the resulting ODoo was 1. The The higher the concentration of Zeocin R) in the YPDS agar BMMY medium (Invitrogen) pH 3.0 contains methanol plates, the more resistance protein has to be produced by the (Mallinckrodt Baker B. V.) which induces the expression of cell in order to quantitatively bind Zeocin R) and thus allow the complete or shortened recombinant trypsinogen via the growth. This is among others possible when multiple copies AOX1 promoter. of the resistance gene are integrated into the genome. Clones 0064. The shaking flasks were incubated at 30°C. while were reinoculated on raster MD plates as described above. shaking, samples were taken every 24 hours, the ODoo was Subsequently these clones were in turn checked for trypsi determined, an aliquot was taken to check the expression of nogen expression and secretion as described above. the complete and shortened recombinant trypsinogen by 0069. Surprisingly it was possible after this measure to means of SDS polyacrylamide gel electrophoresis and each identify clones having considerably increased expression was supplemented with 0.5% methanol (Mallinckrodt Baker yield of the shortened recombinant trypsinogen secreted into B. V.) for further induction. The expression experiments the culture supernatant after SDS polyacrylamide gel elec were carried out for 72 hours. trophoresis. Analysis of the Expression of the Complete and Shortened Recombinant Trypsinogen by Means of SDS Gel Electro Example 5 phoresis Increasing the Expression Yield by Using a Second Selec 0065 500 ul were taken from each expression culture, the tion Pressure ODoo was determined and the cells were centrifuged. The 0070 Increasing the Zeocin R) concentration above 2000 culture Supernatant was stored and the cell pellet was ug/ml did not lead to an improved expression yield of the resuspended for the lysis in an appropriate amount of shortened recombinant trypsinogen. In order to further Y-PERTM (50 to 300 ul/Pierce) for the ODoo and shaken for increase the gene copy number in the expression clones of 1 hour at room temperature. Subsequently the lysate was the gene according to SEQ ID NO. 22 which codes for the centrifuged to separate cell debris (15000xg/5 minutes) and recombinant shortened trypsinogen and is codon-optimized the supernatant was transferred to fresh reaction vessels. 10 for expression in yeast, additional expression vectors were ul lysate and 10 Jul of the culture Supernatant were applied to integrated into the genome of the expression clones prepared an SDS polyacrylamide gel and the proteins were separated in examples 3 and 4 having the highest expression yield by according to size by applying an electrical field. means of a second selection pressure, preferably G418 0.066 Surprisingly it was possible to identify weak bands (Roche Diagnostics GmbH). in the culture Supernatants of clones containing the complete 0071 For this purpose a part of the expression cassette recombinant trypsinogen as well as of clones containing the from pTRYP-11 consisting of a part of the AOX1 promoter, shortened recombinant trypsinogen that did not occur in the the gene for the signal peptide of the C. factor of Saccha control clones. Control clones are to be understood as Pichia romyces cerevisiae, the codon-optimized gene for the pastoris X33 cells that have been transformed with the recombinant shortened trypsinogen according to SEQ ID starting vector pPICZOA and that have been grown and NO.22, is cut out with the restriction endonucleases SacI and induced analogously to the expression clones for trypsino Xbal from pTRYP-11, the restriction mixture is separated on US 2008/0064084 A1 Mar. 13, 2008 a 1% agarose gel and the 1693 bp fragment is isolated from by means of an expanded bed chromatography (McCornick the gel (QIAquick Gel Extraction Kit/Qiagen). At the same (1993); EP 0 699 687) using a cation exchanger (e.g. time the vector pPIC9K (Invitrogen) was cleaved with SacI Streamline(RSP, XL). In this case the chromatography is and NotI, the restriction mixture was separated on a 1% carried out in the presence of the cells. The cells are agarose gel and the 8956 bp vector fragment was isolated simultaneously separated by the chromatography step. Sub from the gel (QIAquick Gel Extraction Kit/Qiagen). The sequently the procedure is as described in example 6.1 Xbal overhang of the fragment from pTRYP-11 and the NotI (rebuffering/activation etc.). overhang from pPIC9K was filled up with Klenow poly merase (Roche Diagnostics GmbH) to form blunt ends Example 7 according to the manufacturers instructions. Subsequently the two fragments obtained in this manner were ligated as Activity Determination described above. The ligation mixture was transformed in E. 0077. The activity of trypsin was determined using Chro coli XL 1 Blue (Stratagene) as described above (the clones mozym TRY (Pentapharm Ltd) in 100 mM Tris pH 8.0, 20 containing plasmid were selected by 100 ug/ml amplicillin in mM CaCl at 25° C. The photometric measurement is the nutrient plates) and checked by means of restriction carried out at 405 nm. analysis and sequencing. The expression vector formed in this way was named pTRYP-13 (see FIG. 3). 0078. Abbreviations: 0072 The integration of the expression vectorpTRYP-13 into the genome of Pichia pastoris was selected by means of YPD: yeast peptone dextrose G418 (Roche Diagnostics GmbH). YPDS: yeast peptone dextrose sorbitol BMGY: buffered glycerol-complex medium 0073. The clones having the highest trypsinogen expres BMMY: buffered methanol-complex medium sion yield from the multiple transformation with pTRYP-11 SDS: Sodium dodecyl sulfate (Zeocin resistance) were prepared for electroporation as described above and transformed with 1 lug of the vector fragment from pTRYP-13 (G418 resistance) linearized with SalI (Roche Diagnostics GmbH). The transformation mix LIST OF LITERATURE REFERENCES ture was subsequently stored for 1 to 3 days at 4°C. in 1 M 0079 Bricteuz-Gregoire, Schyns R., Florkin M (1966) sorbitol (ICN) (for the formation of G418 resistance), then Biochim. Biophys. Acta 127: pp. 277. 100 to 200 ul was plated out on YPD plates (Invitrogen) containing 1, 2 and 4 mg/ml G418 (Roche Diagnostics 0080 Calmels T. Parriche M. Durand H., Tiraby G. GmbH) and incubated for 3 to 5 days at 30° C. The clones (1991), Curr. Genet. 20: pp. 309. resulting therefrom preferably from the YPD plates with the 0081 Charles M., Rovery M., Guidoni A. Desnuelle P. highest G418 concentration, were again examined for an (1963) Biochim. Biophys. Acta 69: pp 115-129. increased expression of the shortened recombinant trypsi nogen using SDS polyacrylamide gel electrophoresis as 0082) Desnuelle P. (1959) The Enzymes 2" Edition vol 4 described above. Editor Boyer, Acad. Press NY. pp. 119. 0074. After this process is was surprisingly possible to 0.083 Drocourt, D., Calmels T. Reynes J. P. Baron M., again identify clones having an increased expression yield of Tiraby G. (1990), Nucleic Acid Research 18: pp. 4009. the shortened recombinant trypsinogen in the culture Super 0084 Graf L., Craik C. S., Patthy A., Roczniak S., natant after SDS polyacrylamide gel electrophoresis. Fletterick R.J., Rutter W.J. (1987) Biochem. 26: pp. 2616. Example 6 0085 Graf L., Jancso A. Szilagyi L., Hegyi G., Pinter K. Naray-Szabo G., Hepp J., Mehzihradszky K. Rutter W. J. Isolating Trypsinogen From the Culture Supernatant and Activation 6.1 (1988) Proc. Natl. Acad. Sci USA 85, pp. 4961. 0075. The culture supernatant was separated from the 0086) Hanahan (1983) J. Mol. Biol. 166: pp 557. cells by microfiltration, centrifugation or filtration. The 0087 Higaki J. N. Evnin L. B., Craik C. S. (1989) trypsinogen was purified by chromatography on phenyl Biochem. 28: pp. 9256. Sepharose fast flow (Pharmacia). The chromatography was carried out in a pH range of 2-4. Autocatalytic activation was 0088. Hedstrom L., Szilagyi L., Rutter W. J. (1992) started by rebuffering the pH to 7-8 in the presence of 20 Science 255: pp 1249. mM CaCl. This autocatalytic activation can be terminated 0089. Jurasek L., Fackre D. Smillie L. B. (1969) Bio again by changing the pH back into the range of 2-4. Active chem. Biophys. Res. Commun 37: pp. 99. trypsin was purified by chromatography on benzamidine Sepharose (e.g. SP-Sepharose(RXL, ff) (Pharmacia/package 0090 Keil B. (1971) The Enzymes Vol II, 3" Edition, insert) or on an ion exchanger. Trypsin is stored at pH 1.5-3 Editor Boyer, Acad. Press N.Y. pp. 249-275. in order to avoid autolysis. The specific activity of the 0.091 Light A., Savithari H. S., Liepnieks J. J. (1980) purified trypsin is 180-200 U/mg protein. Analytical Biochemistry 106: pp 199-206. 6.2 0092 McComick, D. K., Bio/Technol. 11 (1993), 1059: 0.076 The entire fermentation broth is diluted in a ratio of Expanded Bed Absorption, Principles and Methods, about 1:2 to 1:4 with ammonium acetate buffer (5-20 mM) Amersham Pharmacia Biotech, Edition AB, ISBN containing 5-30 mM calcium chloride, pH 3.5 and purified 91-630-551.9-8. US 2008/0064084 A1 Mar. 13, 2008

0093 Morihara K. and TSZZuki J. (1969) Arch Biochem 01.05 WO 97/00316 (NOVO NORDISK AS/Wöldike Biophys 126: pp. 971. Helle, Kjeldsen Thomas) A PROCESS FOR PRODUC 0094) Northrop J. H. Kunitz M., Herriott R. (1948) ING TRYPSIN. Crystalline Enzymes, 2" Edition, Columbia Univ. Press 01.06 WO 99/10503 (ROCHE DIAGNOSTICS/Ko NY. petzki Ehrhard, Hopfner Karl-Peter, Bode Wolfram, 0.095 Ryan C. A. (1965) Arch Biochem Biophys 110: pp Huber Robert) ZYMOGENIC PROTEASE PRECUR 169. SOR THAT CAN BE AUTOCATALYTICALL ACTI 0096 Sambrook, J., Fritsch E. F., Maniatis T. (1989) In. VATED AND THEIR USE. Molecular Cloning: A Laboratory Manual. Cold Spring 01.07 WO 00/17332 (ELI LILLY AND COMPANY/ Harbor Laboratory Press, Cold Spring Harbor, N.Y. Handuier Jose Michael, Hershberger Charles Lee, 0097 Travis J. (1968) Biochem Biophys Res Commun. Desplancq Dominique, Larson Jeffrey) PRODUCTION 30: pp 730. OF SOLUBLE RECOMBINANT TRYPSINOGEN 0098 Travis J. and Roberts R. C. (1969) Biochemistry 8: ANALOGS pp. 2884. 01.08 WO 01/55429 (POLYMUN SCIENTIFIC IMMU 0099 Trop M. and Birk Y. (1968) Biochem. J. 109: pp NBIOLOGISCHE FORSCHUNG GMBHAMattanovich 475. Diethard, Katinger Hermann, Hohenblum Hubertus, 0100 Wahlby S. and Engström L. (1968) Biocheim. Naschberger Stefan, Weik Robert) METHOD FOR THE Biophys. Acta 151: pp. 402. MANUFACTURE OF RECOMBINANT TRYPSIN. 0101 Vasquez, J. R. Evnin L. B., Higaki J. N. Craik C. 01.09 EP 0 597 681 (ELI LILLY AND COMPANY/ S. (1989) J. Cell. Biochem. 39: pp. 265. Greaney Michael Gerard, Rostech Paul Robert) EXPRES SION VECTORS FOR THE BOVINE TRYPSIN AND 0102) Wahlby S. (1968) Biochim. Biophys. Acta 151: pp TRYPSINOGEN AND HOST CELLSTRANSFORMED 394. THEREWITH. 0103) Willett, W. S., Gillmor S.A., Perona J. J., Fletterick 0110 EP 0699 687 (MITSUBISHI PHARMA CORPO R. J., Craik C. S. (1995) Biochem. 34: pp. 2172. RATION/Noda Munehiro, Sumi Akinori, Ohmura Takao, 0104 Yee, L. and Blanch, H. W., (1993) Biotechnol. Yokoyama Kazmasa) PROCESS FOR PURIFYING Bioeng. 41: pp. 781-790. RECOMBINANT HUMAN SERUMALBUMIN.

SEQUENCE LISTING

<16 Oc NUMBER OF SEO ID NOS: 23

<210 SEQ ID NO 1 <211 LENGTH: 247 &212> TYPE: PRT <213> ORGANISM: Porcine

<4 OO SEQUENCE: 1 Ile Pro Asn. Thir Phe Val Lieu. Lieu Ala Lieu. Lieu. Gly Ala Ala Wall Ala 1. 5 1O 15 Phe Pro Thr Asp Asp Asp Asp Llys Ile Val Gly Gly Tyr Thr Cys Ala 2O 25 3 O

Ala Asn Ser Ile Pro Tyr Glin Val Ser Lieu. Asn Ser Gly Ser His Phe 35 4 O 45

Cys Gly Gly Ser Lieu. Ile Asn Ser Gln Trp Val Val Ser Ala Ala His SO 55 60 Cys Tyr Lys Ser Arg Ile Glin Val Arg Lieu. Gly Glu. His Asn. Ile Asp 65 70 7s

Val Lieu. Glu Gly Asn. Glu Glin Phe Ile Asn Ala Ala Lys Ile Ile Thr 85 90 95

His Pro Asn. Phe Asin Gly Asn. Thir Lieu. Asp Asin Asp Ile Met Lieu. Ile 1OO 105 110

Llys Lieu. Ser Ser Pro Ala Thr Lieu. Asn. Ser Arg Val Ala Thir Wal Ser 115 12O 125 US 2008/0064084 A1 Mar. 13, 2008

- Continued Lieu Pro Arg Ser Cys Ala Ala Ala Gly Thr Glu. Cys Lieu. Ile Ser Gly 13 O 135 14 O Trp Gly Asn Thr Lys Ser Ser Gly Ser Ser Tyr Pro Ser Lieu. Leu Gln 145 150 155 160 Cys Lieu Lys Ala Pro Val Lieu. Ser Asp Ser Ser Cys Llys Ser Ser Tyr 1.65 17O 17s Pro Gly Glin Ile Thr Gly Asn Met Ile Cys Val Gly Phe Lieu. Glu Gly 18O 185 19 O Gly Lys Asp Ser Cys Glin Gly Asp Ser Gly Gly Pro Val Val Cys Asn 195 2OO 2O5 Gly Glin Lieu. Glin Gly Ile Val Ser Trp Gly Tyr Gly Cys Ala Glin Lys 21 O 215 22O Asn Llys Pro Gly Val Tyr Thr Llys Val Cys Asn Tyr Val Asn Trp Ile 225 23 O 235 24 O

Glin Glin. Thir Ile Ala Ala Asn 245

<210 SEQ ID NO 2 <211 LENGTH: 744 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic gene <4 OO SEQUENCE: 2 attic caaata ctitttgttitt gttggctttgttgggtgctg. citgttgctitt to caact gat 6 O gatgatgata aaattgttgg toggittatact tdtgctgcta attct attcc at atcaagtt 12 O t ctittaaatt ctdgttct catttttgtggt gigttctittga ttaattctica atgggttgtt 18O tctgctgctic attgttacaa atcaagaatc caagttagat tdggtgaaca taatattgat 24 O gttittggaag gtaatgaaca atttattaat gctgctaaaa ttatt actica tocaaattitt 3OO aatggtaata ctittggataa tdatattatgttgattaaat tdt cittctic c agctactitta 360 aattcaagag ttgctact.gt ttctittgcca agat cittgtg citgctgctgg tactgaatgt 42O ttgatttctg gttggggtaa tactaaatct tctggttctt cittatccatc tttgttgcaa. 48O tgtttgaaag ctic cagttitt gttctgattct tcttgtaaat citt cittaccc aggtoaaatt 54 O actggtaata tatttgttgt tdtttitttg galaggtggta aagatt Cttg tca aggtgat 6OO tctggtggtc. Cagttgtttg taatggtcaa ttgcaaggta ttgtttcttggggittatggit 660 tgtgct caaa aaaataaacc aggtgtttac actaaagttt gtaattatgt taattggatt 72 O caacaaacta ttgctgctaa ttag 744

<210 SEQ ID NO 3 <211 LENGTH: 56 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 3 gcqgaattica ttccaaatac titttgttttgttggctttgttgggtgctgc tigttgc 56

<210 SEQ ID NO 4 <211 LENGTH: 54 &212> TYPE: DNA US 2008/0064084 A1 Mar. 13, 2008 10

- Continued <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 4 ccaccaacaa tttitat catc atcat cagtt ggaaaagcaa cagcago acc caac 54

<210 SEQ ID NO 5 <211 LENGTH: 62 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 5 gatgataaaa ttgttggtgg ttatacttgt gctgctaatt ctatt coata t caagtttct 6 O tt 62

<210 SEQ ID NO 6 <211 LENGTH: 64 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 6 gaattaatca aagaaccacc acaaaaatga gaaccagaat ttaaagaaac ttgatatgga 6 O atag 64

<210 SEQ ID NO 7 <211 LENGTH: 64 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 7 ggtggttctt tdattaattic ticaatgggitt gtttctgctg ct cattgtta caaatcaaga 6 O at CC 64

<210 SEQ ID NO 8 <211 LENGTH: 62 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 8 cct tccaaaa cat caatatt atgttcaccc aatctaactt ggatt cittga tttgtaacaa 6 O tg 62

<210 SEQ ID NO 9 <211 LENGTH: 63 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 9 taat attgat gttittggaag gtaatgaaca atttattaat gctgctaaaa ttatt actica 6 O US 2008/0064084 A1 Mar. 13, 2008 11

- Continued t cc 63

<210 SEQ ID NO 10 <211 LENGTH: 62 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 10 caacataata t cattatcca aag tattacc attaaaattt ggatgagtaa taattittagc 6 O ga 62

<210 SEQ ID NO 11 <211 LENGTH: 64 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 11 ctittggataa tdatat tatgttgattaa at tdt cittct co agctactitta aattcaagag 6 O ttgc 64

<210 SEQ ID NO 12 <211 LENGTH: 58 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 12

Ccagcagcag cacaagat ct tcaaagaa acagtagcaa citcttgaatt taaagtag 58

<210 SEQ ID NO 13 <211 LENGTH: 61 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 13

Cttgttgctgc tigctgg tact gaatgtttga tittctggttgggg taatact aaatcttctg 6 O

9 61

<210 SEQ ID NO 14 <211 LENGTH: 58 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 14 gagctittcaa acattgcaac aaagatggat aagaagaacc agaagattta gtatt acc 58

<210 SEQ ID NO 15 <211 LENGTH: 60 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer US 2008/0064084 A1 Mar. 13, 2008 12

- Continued <4 OO SEQUENCE: 15 gttgcaatgt ttgaaagctic cagttttgtc. tdatt cittct totaaatctt cittacccagg 6 O

<210 SEQ ID NO 16 <211 LENGTH: 59 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 16 c caaaaaacc aacacaaatc at attaccag taatttgacc toggg taagaa gatttacaa 59

<210 SEQ ID NO 17 <211 LENGTH: 60 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 17 gatttgttgtt ggtttitttgg aaggtggtaa agatt Cttgt Caaggtgatt CtggtggtcC 6 O

<210 SEQ ID NO 18 &2 11s LENGTH: 57 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 18 c caagaaa.ca ataccttgca attgaccatt acaaacaact ggaccaccag aat cacc f

<210 SEQ ID NO 19 <211 LENGTH: 51 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 19 gcaagg tatt gtttcttggg gtt atggttgtctcaaaaa aataalaccag g 51

<210 SEQ ID NO 2 O <211 LENGTH: 90 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 2O gcqtctagac taattagcag caatagtttgttgaatccaa tta acataat tacaaactitt 6 O agtgtaaa.ca cct ggttt at ttttittgagc 9 O

<210 SEQ ID NO 21 <211 LENGTH: 48 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: &223> OTHER INFORMATION: Primer

<4 OO SEQUENCE: 21 US 2008/0064084 A1 Mar. 13, 2008 13

- Continued ccgggaattic gatgatgatg ataaaattgt ttggittat acttgttgc 48

<210 SEQ ID NO 22 <211 LENGTH: 687 &212> TYPE: DNA <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Synthetic gene <4 OO SEQUENCE: 22 gatgatgatgataaaattgt toggtggittat acttgttgctg ctaattictat tccatat caa 6 O gtttctittaa attctggttct catttttgt gigtggttctt tdattaattic ticaatgggitt 12 O gtttctgctg ct cattgtta caaatcaaga atccaagtta gattgggtga acataatatt 18O gatgttittgg aaggtaatga acaatttatt aatgctgcta aaatt attac to atccaaat 24 O tittaatggta at actittgga taatgatatt atgttgatta aattgtc.ttic ticcagctact 3OO ttaa attcaa gagttgctac tdtttctittg ccaagat citt gtgctgctgc tigg tactgaa 360 tgtttgattt ctdgttgggg taatactaaa tottctggitt citt cittatcc atc.tttgttg 42O caatgtttga aagctic cagt tttgtctgat t cittcttgta aatcttctta cccaggt caa 48O attactggta atatgatttgttgttggtttt ttggalaggtg gtaaagattic ttgtcaaggt 54 O gattctggtg gtc.cagttgt ttgtaatggit caattgcaag gtattgttt C ttggggittat 6OO ggttgttgctc aaaaaaataa accaggtgtt tacactaaag tttgtaatta tottaattgg 660 attcaacaaa citattgctgc taattag 687

<210 SEQ ID NO 23 <211 LENGTH: 228 &212> TYPE: PRT <213> ORGANISM: Artificial sequence &220s FEATURE: <223> OTHER INFORMATION: Shortened Trypsinogen <4 OO SEQUENCE: 23 Asp Asp Asp Asp Llys Ile Val Gly Gly Tyr Thr Cys Ala Ala Asn. Ser 1. 5 1O 15 Ile Pro Tyr Glin Val Ser Lieu. Asn Ser Gly Ser His Phe Cys Gly Gly 2O 25 3O Ser Lieu. Ile Asn. Ser Glin Trp Val Val Ser Ala Ala His Cys Tyr Lys 35 4 O 45 Ser Arg Ile Glin Val Arg Lieu. Gly Glu. His Asn. Ile Asp Val Lieu. Glu SO 55 6 O Gly Asn. Glu Glin Phe Ile Asn Ala Ala Lys Ile Ile Thr His Pro Asn 65 70 7s 8O Phe Asin Gly Asn. Thir Lieu. Asp Asin Asp Ile Met Lieu. Ile Llys Lieu. Ser 85 90 95 Ser Pro Ala Thir Lieu. Asn. Ser Arg Val Ala Thr Val Ser Lieu Pro Arg 1OO 105 11 O

Ser Cys A a. Ala Ala Gly Thr Glu. Cys Lieu. Ile Ser Gly Trp Gly Asn 115 12 O 125 Thr Lys Ser Ser Gly Ser Ser Tyr Pro Ser Lieu. Leu Gln Cys Lieu Lys 13 O 135 14 O Ala Pro Val Lieu Ser Asp Ser Ser Cys Llys Ser Ser Tyr Pro Gly Glin 145 150 155 160 US 2008/0064084 A1 Mar. 13, 2008 14

- Continued Ile Thr Gly Asn Met Ile Cys Val Gly Phe Leu Glu Gly Gly Lys Asp 1.65 17s

Ser Gln Gly Asp Ser Gly Gly Pro Wall Wall Cys Asn Gly Gln Lieu. 18O 185 19 O

Glin Gly Ile Wall Ser Trp Gly Tyr Gly Cys Ala Glin Lys Asn Llys Pro 195 2O5

Gly Wall Tyr Thr Lys Val Cys Asn Tyr Val ASn Trp Ile Glin Gn. Thir 21 O 215

Ile Ala Ala Asn 225

. A trypsinogen protein comprising SEQ ID NO: 1 bound to the N-terminus of the amino trypsin sequence and a shortened propeptide, said acid sequence of trypsin, and wherein said signal propeptide consisting of no more than 10 amino acids, peptide is bound to the N-terminus of said propeptide wherein the propeptide retains the amino acid sequence and mediates secretion of trypsinogen from the host from position 20 to position 25 of SEQID NO: 1 bound cell, to the N-terminus of the amino acid sequence of b) culturing the yeast host cell in a first culture medium trypsin. under conditions promoting the growth of the yeast 2. The trypsinogen protein of claim 1 further comprising host cell; a signal peptide, wherein said signal peptide is bound to the c) culturing the yeast host cell in a second culture medium N-terminus of said propeptide and mediates secretion of having a pH of from 3.0 to 4.0 and inducing the trypsinogen from a yeast host cell. expression of said recombinant nucleic acid, whereby 3. The trypsinogen protein of claim 1 wherein said the yeast host cell expresses trypsinogen comprising propeptide sequence comprises an enterokinase recognition the shortened propeptide in the second culture medium site. significantly more than trypsinogen comprising a com 4. The trypsinogen protein of claim 3 wherein said plete propeptide, propeptide sequence consists of an enterokinase recognition site having the amino acid sequence Asp-Asp-Asp-Asp-Lys d) isolating the trypsinogen comprising the propeptide and optionally up to 5 additional amino acids located from the second culture medium N-terminally thereof. 8. A nucleic acid encoding the trypsinogen peptide of 5. The trypsinogen protein of claim 4 wherein said trypsin claim 1. sequence is a porcine trypsin amino acid sequence. 9. The nucleic acid of claim 8 wherein the nucleic acid 6. The trypsinogen protein of claim 1, wherein the nucleic comprises the nucleotide sequence shown in SEQ ID NO. acid comprises the amino acid sequence shown in SEQ ID 22. NO. 23. 10. The nucleic acid as claimed in claim 9, wherein the 7. A trypsinogen protein produced by the steps comprising nucleic acid is operatively linked to a regulatable expression control sequence. a) preparing a yeast host cell comprising a recombinant 11. A recombinant host cell, wherein the cell is trans nucleic acid that encodes a trypsinogen protein com formed with the nucleic acid of claim 8. prising a signal peptide and a shortened propeptide 12. The recombinant host cell of claim 11 wherein Pichia sequence, said propeptide consisting of no more than pastoris or Hansenula polymorpha is used as the host cell. 10 amino acids, wherein the propeptide retains the amino acid sequence from position 20 to position 25 of k k k k k