Field of Invention

The invention relates to Pichia pastoris strain bearing a DNA expression cassette integrated in its genome, also to the DNA expression cassette, and to a method for cultivation of said Pichia pastoris yeast for the production of human recombinant enterokinase as a soluble protein.

Invention Background

Enterokinase (EC 3.4.21.9) is the serine protease of the intestinal brush border membrane. From the physiological point of view the enterokinase activates its native substrate trypsinogen to trypsin by the digestion of N-terminal part of the peptide, after which the conservative sequence of four aspartate acids and one lysine (Asp)4-Lys (Lu, 1997) is situated. It is this five amino acids sequence, which is specifically recognized by enteropeptidase cutting off the N-terminal protein from C-terminal one immediately after this amino acid sequence. The separating of the N-terminal part results in active trypsin, which subsequently activates many other pancreatic zymogens (Kunitz, 1939). The native enteropeptidase dimer was isolated from porcine (Matsushima et at. , 1994), bovine (Kitamoto, 1994), murine (Yuan et at. , 1998), rat (Yahagi, 1996), fish (Japanese rice fish) (Ogiwara and Takahashi, 2007), and human (Kitamoto et al. , 1995) intestine. In all cases the enteropeptidase appears to be a dimer having the chains interconnected by a disulfide bond. The enteropeptidase proenzyme is a single-strand polypeptid consisting of heavy (82-140 kDa) and light (35-62 kDa) chains. The heavy chain comprising N-terminal membrane domain only slightly influences the recognition of the digestion site in small peptides, but strongly influences the recognition of a substrate, if the substrate has macromolecular character, and also strongly influences inhibitor specificity (Lu et al., 1997). The light chain is the catalytic subunit of enteropeptidase and comprises serine protease chymotrypsine like domain (Lu and Sadler, 1998).

High specificity level of the enterokinases makes this enzyme ideal for the digestion of the affinity labels or fusion proteins, and thus their distinguishing from the target recombinant proteins produced in bacteria. The expression and purification of the recombinant catalytic unit of bovine enteropeptidase were described for various host strains, such as Escherichia coli (Collins-Racie et at., 1995; Yuan and Hua, 2002; Tan et at., 2007), yeast Saccharomyces cerevisiae (Choi et at., 2001 ), methylotrophic yeast Pichia pastoris (Vozza et at., 1996), filamentous fungus Aspergillus niger (Svetina et at., 2000), and for COS-1 cells from monkey kidney (Vallie et at., 1993).

Furthermore, the human enteropeptidase was also expressed in E. coli cells. The peptidase was expressed in the fusion with thioredoxine in the form of inclusion bodies, and was renaturated by a dilution method. The dilution renaturation enabled to obtain maximally only 2 % of the active peptidase, but the activity of its active portions was about 5-times higher than in the recombinant bovine enteropeptidase obtained from yeast P. pastoris. It is confirmed by the measured values of K _m a k _ca t constants of this enzyme (Gasparian et at. , 2003). The results of the enterokinase expression in E. coli cells published by different authors are very inconsistent. While Gasparian (2003) et al., were able to refold human enterokinase in about 2 % of the inclusion bodies, even if at temperature of 37 °C, there was more than 90 % of the product in the form of the inclusion bodies, Liu and Hua (2002) expressed the bovine enzyme in the same vector, at the same temperature in the form of the soluble and active protein with the specificity constant approximately the same as those measured by Gasparian et al. (2003) for bovine enteropeptidase expressed in P. pastotris. It is necessary to say that Gasparian et al. (2003) observed no activity in the soluble faction. The enteropeptidase was expressed also fused with glutation S-transferase, wherein 90 % of the product was in the form of the inclusion bodies. The total amount of the obtained mature enterokinase after the purification, refolding and autokatalytic cleavage of the enzyme was 27.5 mg/1 I of the fermentation culture. The activity of the purified enteropeptidase was comparable with the activity of the commercial enterokinase provided by Sigma (Tan et al., 2007). The bovine enterokinase was for the first time produced in P. pastoris in 1996 (Vozza et al., 1996). After purification, the obtained net yield of the pure peptidase was 6.3 mg.h ¹ of the fermentation medium, with the specific activity of 168 U.m . It was higher value than those achieved by the expression in E. coli (Collins-Racie et al. , 1995).

In 2004 (Fang et al.) an attempt to produce the bovine enterokinase in P. pastoris cells under control of pAOX promoter was done. The amount of the secreted bovine enterokinase in medium was 10 mg.h ¹ of the fermentation culture. For the improvement of the enzyme purification in the subsequent, downstream step, His-tag was attached to the DNA sequence encoding the enterokinase. The enterokinase yield achieved the value of 5.4 mg.l ^"1 of the fermentation medium with the activity of 80 U.mg- ¹ (Fang et al. , 2004) representing even lower yield as reported in the previous publication (Vozza et al.1996). The bovine enteropeptidase obtained by such a way was able to digest almost 100 % of the substrate in 16 hours at temperature of 16 °C (Fang et al., 2004), when a ratio of the substrate, i.e. the fusion protein GST-vasostatinin with the cleavage site for bEK between its parts, to enterokinase enzyme was 1000:1 . The subsequent research was focused on the increasing of the yield of bovine recombinant enterokinase via monitoring of the impact of the selected strain (methanol utilizing phenotype of the host organism), via optimization of the fermentation conditions considering the influence of pH value during the cultivation, as well as the influence of the used carbon source, on the expression level. Upon using of the strains having the methanol induced promoters under the control of pAOX promoter, strain MutS showed to be more suitable for the production of the recombinant bovine enterokinase than strain Mut+. The advantage of MutS strain is its reduced sensitivity to residual methanol in the cultivation medium in comparison to Mut+ strain. After 120-hour induction in the medium with glycerol and methanol, at pH6, the obtained expression value with enterokinase production was 350 mg.l ^"1 of the fermentation medium. After the purification 150 mg of the purified enzyme/I of the medium with specific activity of 9000 U (international enzyme activity unites). mg- ¹ were obtained (Peng et al. , 2004). This activity was two-times lower than those of the enterokinase produced by the filamentous fungus A. niger (Svetina et al. , 2000). After the attachment of His-tag to the C-terminus of the gene encoding the bovine enterokinase the specific enzyme activity was reduced to 8,000 U. mg- ¹ (Peng et al. , 2004).

The heterogeneous protein production rate depends, apart from the conditions and parameters of the cultivation process, also on the selection of the promoter as discussed in Pepeliaev et al. , (201 1 ). In some cases upon constitutive expression of the recombinant proteins with pGAP promoter higher production is achieved than upon the classic expression under the control of the inducible pAOX promoter. The employing of the construct using pGAP promoter possesses some disadvantages. One of such disadvantages is the exclusion of methanol as inductor and carbon source during fed- batch cultivation, which results in the reduction of cell lysis and thus the reduction of the proteolytic activity of the secreted proteases (Zhang et ai , 2009). By the comparison of the human enterokinase expression rates upon using of various promoters the highest expression rate was observed in the experiments using pGAP promoter. After 120 hours of the cultivation the observed enterokinase concentration was 73.5 mg.h ¹ of the cultivation medium with activity of 20,000 U.I ^"1 . The production rate was recalculated on the basis of the specific activity of the commercial bovine enterokinase EKMax® (Invitrogen), showing three-times lower activity than the activity of the human recombinant enterokinase.

The production of the enteropeptidase in the Aspergillus niger expression system was described in Svetina et al. (2000). They expressed cDNA encoding catalytic subunit of the bovine enterokinase fused with linker protein glucoamylase 2. After the purification via the ion exchange chromatography they obtained the yield of 1 mg.h ¹ of the cultivation media of the high-activity enzyme, enteropeptidase, having higher specific activity, 19,880 U.mg- ¹ than commercial EKMax® (Invitrogen) (Svetina et ai, 2000).

US 2015/0020238 A1 relates to a plant cell transformed by a recombinant vector comprising a synthetic gene encoding the human enterokinase light chain. Here, rice is used as the plant cell.

WO2012071257 (US 8 557 558) describes the preparation of polynucleotide molecules encoding bovine enterokinase, the yeast expression construct comprising the yeast expression vector and polynucleotide molecules encoding bovine enterokinase, the yeast cells comprising said expression construct, methods for digestion and preparation of the recombinant polypeptid using bovine enterokinase prepared by such methods.

Disclosure of the invention

Subject matter of the invention is formed by a DNA expression cassette carrying a gene encoding the human enterokinase light chain of SEQ ID NO: 3.

Pursuant to other embodiment the subject matter of this invention is formed by the DNA expression cassette carrying the gene encoding secretion signal, a - Mating Factor from Saccharomyces cerevisiae of SEQ ID NO: 2, which is fused to 5 -end of the gene encoding the light chain of human enterokinase of SEQ ID NO: 3 in the same direction.

Another subject matter of this invention is the DNA expression cassette carrying the promoter of the gene for glyceraldehyde phosphate dehydrogenase of SEQ ID NO: 1 functionally preceding the gene encoding the secretion signal aMF from Saccharomyces cerevisiae of SEQ ID NO: 2, which is fused to the 5 -end of the gene encoding the light chain of human enterokinase of SEQ ID NO: 3 in the same direction.

According to the preferred embodiment of this invention the DNA expression cassette carrying the promoter of the gene for glyceraldehyde phosphate dehydrogenase of SEQ ID NO: 1 functionally preceding the gene encoding the secretion signal aMF from Saccharomyces cerevisiae of SEQ ID NO: 2, which is fused to the 5 -end of the gene encoding the light chain of the human enterokinase of SEQ ID NO: 3 in the same direction, wherein the sequences encoding the proteolytic cleavage sites for proteases encoding by genes Kex2 and Ste13 are situated between the sequence encoding the secretions signal and the gene encoding the human enterokinase light chain of SEQ ID NO: 3, wherein the human enterokinase light chain, after proteolytic treatment via the cleavage at its N- terminus, comprises only the amino acid residues belonging to the human enterokinase light chain. The subject matter of this invention is formed also by the high-production Pichia pastoris strain carrying the above described DNA expression cassette integrated in its genome.

Further, the subject matter of this invention is formed also by a method for the cultivation of the high-production Pichia pastoris strain carrying the above described DNA expression cassette integrated in its chromosome for the production of the human enterokinase light chain. The cultivation process is performed in two phases, while in the first growth phase the cultivation conditions are set to keep high specific growth rate of the biomass to at least 0.02 hr ¹ , and in the second production cultivation phase the cultivation conditions are set to keep low specific growth rate of the biomass to maximally 0.01 hr ¹ , while in this cultivation phase the biomass increase is minimized, but the significant production and accumulation of the recombinant human enterokinase in the cultivation medium in the form of soluble protein is achieved.

According to the preferred embodiment the first grow cultivation phase occurs at the beginning of the cultivation to up to the 72 ^nd hour of the cultivation, when the exponential increase of the production strain biomass is observed, which increase is expressed as dry biomass weight. Afterwards the conditions for the second production cultivation phase of the production strain are set during 24 to 36 hours after the 72 ^nd hour of the cultivation, while the second production phase lasts 60 to 80 hours, during which the recombinant human enterokinase is accumulated in the cultivation medium in the form of the soluble protein.

According to the other preferred embodiment, in the first growth phase the conditions for the process control, considering its aeration and mixing, are set by such a way to achieve a high value of partial dissolved oxygen tension (DOT), particularly 55 to 65 % of the saturation, and via the controlled supplementing of biomass by the carbon substrate, in order to ensure the high specific rate of the biomass growth, expressed as dry biomass weight (DCW) in the range from 150 to 200 g/l of the cultivation medium. The subject matter of the invention is the DNA expression cassette consisting of at least the yeast promoter region, for example the promoter of glyceraldehyde phosphate dehydrogenase gene, after which the nucleotide sequence encoding the secretion signal with the nucleotide sequence encoding a place suitable for the proteolytic processing for the cutting off the secretion signal is functionally bound. Furthermore the DNA expression cassette carries the nucleotide sequence encoding the human enterokinase light chain functionally bound after the secretion signal, having 6 histidines positioned after the C- terminus of the enterokinase gene. The sequences included for the separation of the secretion signal from the sequence encoding the human enterokinase light chain are placed by such a way that after the processing no amino acid non-belonging to the human enterokinase light chain remains at N-terminus of the human enterokinase gene.

The subject matter is also the Pichia pastoris yeast itself carrying the described DNA expression cassette integrated in its genome, in one or more copies.

Also the method for the cultivation of modified Pichia pastoris yeast carrying the genetic information encoding the human enterokinase light chain integrated in the chromosome is the subject matter of the invention. This genetic information is functionally bound to the constitutive promoter enabling the expression of said genetic information to the active enzyme form. A secretion signal is the part of the genetic information, which signal enables the expression of the enzyme into the extracellular space, while there is a site for the proteolytic digestion between the secretion signal and the human enterokinase light chain, which place ensures the eliminating of the secretion signal during the secretion pathway without leaving any amino acid residue at N-terminus of the polynucleotide encoding enterokinase. Also the sequence encoding 6 histidines at C-terminus of this polynucleotide for facilitating of the enzyme purification is the part of the genetic information.

The using of the method for the cultivation of the genetically modified Pichia pastoris yeast of the present invention results in the significant increase of the human recombinant enterokinase production in comparison with the available prior art information. The cultivation process is based on two-phase cultivation.

In both cultivation phases the complex supplement comprising glucose as the carbon source is continually added to the cultivation medium, while the feeding rate is adapted to the rate of the culture growth and the technical specification of the fermentation device parameters. The important parameter for the cultivation process control is the maintenance of the partial dissolved oxygen tension (DOT) at the level higher than 30 % of the saturation, because the physiologic processes in P. pastoris yeast run in aerobic modus the most effectively. In the first growth cultivation phase the DOT level is kept by the increasing of the mixing and aeration of the medium. Such regulation can be achieved either manually, or if the fermenter device is equipped with the automatic system for the revolution number increase in relation to the oxygen consumption, it is possible to control this process semi-automatically with the manual increase of the aeration, i.e. via the continual air supply under the medium level. In case of the system equipped both with the multi-level automatic system for the mixing revolution control (Gas Flow Controller) and gas mixing control (Gas Mix), this process can be fully automated. By the automatic gas mixing it is possible to enrich the aerated mixture with oxygen and thus increase not only the effectiveness of the first biomass increase phase, but also the second phase of the human enterokinase production.

During the entire first cultivation phase the feeding process, i.e. the supplying of the carbon source to the solution, is performed with the feeding rate ensuring such specific rate of the biomass growth increase that is closely under the maximal specific growth rate. The feeding rate is controlled by the reaction of the biomass to the oxygen consumption. If during the increase of the feeding rate the cell would not react by the increase of oxygen consumption, the rate is higher or equal to the rate ensuring the maximal specific growth rate. During the first growth cultivation phase the specific growth rate should not decrease under 0.02 hr ¹ , preferably under 0.05 hr ¹ . The first cultivation phase ends either by the obtaining of the specific parameters of the fermentation device, or by the obtaining of such biomass density, at which the specific growth rate starts to decrease quickly and is reduced to the level of 0.01 hr ¹ or under this level. The second production cultivation phase is characterized by a low specific growth rate in the range from 0.01 r ¹ to 0.001 hr ¹ , while the feeding rate is decreased, in order to maintain the maximal obtained mixing, aeration parameters, and the maximal parameters of the gas mixture feeding, i.e. air and oxygen feeding, in the second phase. During this phase the rate of the oxygen consumption is increased in relation to the given feeding rate. In other words, at the constant feeding rate the oxygen consumption by the biomass is increased with time. Therefore, it is necessary to decrease the feeding rate, in order to keep the desired DOT. During this second production phase of the cultivation the enterokinase is significantly accumulated in the medium.

The high cell density cultivation of the Pichia pastoris production strain enables the extending of the cultivation period to 140 - 160 hours. In this second cultivation phase the production of recombinant human enterokinase (rhEK) is significantly increased to the range from 1 ,000 to 3,000 U/ml of the cultivation medium filtrate. This recombinant human enterokinase is excreted from the cells of Pichia pastoris production strain to the medium in the form of the soluble protein.

Brief description of the figures

Figure 1 shows the record of electrophoretic assay of the production ability of the individual clones tested by the bank cultivation.

Figure 2 illustrates the graph of the cultivation profile of P. pastoris strain, when cultivated in the laboratory fermenter by two-phase cultivation.

Figure 3 illustrates the enzyme profile of enterokinase activity in the medium tested during 7-day cultivation (168 hours).

Examples of Invention Embodiment

Example 1 : Preparation of the production strain for rhEKi. production Plasmid pGZE with the gene of zeocine resistance and the promoter from glyceraldehyde dehydrogenase (GAP) gene from Pichia pastoris, after which the gene encoding the polypeptide consisting of "alpha-mating factor" (a MF) as the secretion signal was functionally attached, and the gene of the human enterokinase light chain with his-tag amino acid motive at its C-terminus (HLEKh), was used for the transformation of electro- competent cells of wild-type strain Pichia pastoris Y1 1430. Kex2 protease cleavage site and the target sequences for dipeptidyl aminopeptidase encoded by gene Ste3 are placed between aMF and HLEKh, in order to avoid any amino acid residue non-belonging to the human eterokinase light chain to be present at the N-terminus of the human enterokinase light chain after the proteolytic processing. Before the electroporation the plasmid was linearized by restriction endonuclease Ανή\ and purified by a DNA fragments purification kit. The aliquot of 50 μΙ electrocompetent cells was transformed by the linearized plasmid in total amount of 5 μg and the transformed cells were selected on the solid YPD (1 % yeast autolysate; 2% peptone; 2% glucose) medium supplemented with zeocin (100 μg/ml). The transformats were cultivated in bank with the agitation at selection conditions, at 29 °C 3 days. Approximately 20 colonies, which were the positive transformants, were selected from this medium. These new colonies were reseeded in 2 ml of the liquid cultivation medium YPD supplemented with zeocin and cultivated at 29 °C, 24 hours with agitation.

After the cultivation the chromosomal DNA, for which the presence of the integrated expression plasmid was proved via PCR, was isolated from the selected clones. Almost in all clones it was possible to demonstrate the presence of the expression plasmid. The clones, for which the presence of the plasmid DNA in the chromosome was demonstrated, were tested for the activity of the humane enterokinase enzyme in the cultivation medium without an antibiotic. From 2 ml of the night Pichia pastoris culture from the selected clones in YPD medium 20 μΙ was reseeded to 20 ml of new YPD medium and these were cultivated 144 hours, while 1 ml of 50% solution of sterile glucose were added therein daily. After the end of the cultivation the cells were separated from the medium by centrifugation. The test for human enterokinase activity was performed by the application of the medium from the tested clones to 20 μ9 of the protein substrate carrying the enterokinase cleavage site in 5 μΙ volume in 10 mM Tris-HCI, pH8. The medium was diluted 10 to 100-times (figure 1 ). The digestion effectiveness was assessed by densitometry GeneTools software of SynGene Company. The clone having the best effectiveness was selected for further testing for the enterokinase production by the fermentation in larger volumes.

Figure 1 shows the record of the electrophoretic tests for the productivity of the individual clones in the bank expression. Line 1 : molecule weights standard; line 2: non-cleaved substrate; 3: the substrate cleaved by the commercial enterokinase; lines 4-10: clones with 10x diluted medium; lines 1 1-17: corresponding clones with 100x diluted medium.

Example 2: Cultivation of Pichia pastoris in the laboratory fermenter for production of rhEKL via initial batch phase process

The selected clones according to Example 1 were reseeded to the fresh YPD (1 % yeast autolysate; 2% peptone; 2% glucose) agar plates, and statically cultivated at temperature of 29°C. Afterwards one isolated colony from YPD plate was seeded to 50 ml of the inoculation medium (13.8 g/l (NH ₄ ) ₂ S0 ₄ , 46 g/l (NH ₄ )H ₂ P0 ₄ , 15.9 g/l KOH, 10 g/l glucose; 0.4 ml/l (v/v) 1 M MgS0 ₄ , 0.02 ml/l (v/v) 1 M CaCI ₂ , 4.6 ml/l PTM1 , 10g/I yeast autolysate, 20 g/l peptone) in 250ml Erlenmeyer flask and the culture was cultivated 24 hours at temperature of 29 °C. The solution of microelements and growth substances marked as PTM1 with the following composition was added to the medium: 0.5 g/l C0CI2.6H2O, 65 g/l FeS0 ₄ .7H ₂ 0, 3 g/l MnS0 ₄ .5H ₂ 0, 5 ml/l H ₂ S0 ₄ (95-98%), 0.08 g/l Kl, 6 g/l CuS0 ₄ .5H ₂ 0, 20 g/l ZnCI ₂ , 0.02 g/l H3BO3, 0.2 g/l Na ₂ Mo0 ₄ .2H ₂ 0, and 0.2 g biotin.

The batch fermentation was carried out in the laboratory fermenter in total volume of 2 liters, at temperature of 29 °C, while 750 ml of the following medium was used for the fermentation: 26.7 ml/l H ₃ P0 ₄ , 5.6 ml/l H ₂ S0 ₄ , 15.9 g/l (w/v) KOH, 10 g/l yeast autolysate and 20 g/l peptone. After the sterilization the medium was supplemented with 4.6 ml/l PMT1. The batch medium consisted of 500 g/l glucose, 100 mM MgS0 ₄ , 5mM CaS0 ₄ , and 12 ml/l PMT1. The cultivation conditions were controlled and maintained as follows: pH value of 6.0 via 30% ammonium hydroxide, partial dissolved oxygen tension (DOT) in the medium was kept at 60 % of its saturation value via automatic increasing of the agitator revolutions, further via manual adaptation of the volume in the air hoses for gases supplying into the cultivation media per time unit (vvm = volume of aeration per volume of medium per 1 minute), as well as via manual dispensing of the supplementing medium according to the specific biomass grow rate in the medium.

Upon obtaining of all desired parameters, at the beginning of the cultivation 30 ml of the feeding medium was supplied to the fermenter and 50 ml of inoculum from 24-hour cultivation by the inoculation flask and air pressure via the Pichia pastoris strain carrying the gene encoding the human enterokinase light chain, as described in detail above, in its chromosome, was inoculated to the fermenter. After the consumption of the carbon source contained in the basic cultivation medium, the continual supply of the feeding solution comprising the identical carbon source was initiated, while the feeding rate was adapted to the cell growth rate. Feeding rate had been increasing until the cells reacted to the increasing of the carbon substrate concentration in the medium by the increasing of oxygen consumption. After the obtaining of such feeding supply rate, at which the cells had stopped to react to the higher supply and subsequently to the increased concentration of the carbon source in the medium, the feeding rate was reduced to the last value, at which the culture still reacted by the increased consumption of dissolved oxygen. With the increasing need of oxygen the mixing revolutions were increasing automatically, and aeration was increasing manually. The aeration was increasing by such a way that at 500 rpm (revolution per minute) the aeration was set to 2 vvm, at 1000 rpm to 3 vvm, and at 1500 rpm to 4 vvm. By this method the specific rate of the biomass growth was kept above 0.05 h ^"1 until obtaining the revolutions of 2,000 rpm, and the aeration of 4 vvm. This phase lasted from hour 40 to hour 60 of the cultivation.

Upon obtaining of these cultivation parameters (Figure 2), the second phase of the culture growth followed, during which the feeding was decreasing by such a way to reduce the specific growth rate under 0.01 h ^~1 , while the biomass growth was rather slower in this phase, in comparison with the first phase (Figure 2). The second cultivation phase lasted from hour 70 to hour 80. In this phase the considerable accumulation of the human recombinant enterokinase (rhEK) in the medium in the form of soluble protein occurred. The highest concentrations were measured from hour 144 to hour 168 of the cultivation (Figures 2, 3). rhEK activity in the fermented medium with Pichia pastoris production strain was tested using the commercial substrate Trx-DCD1 carrying the specific enterokinase cleavage site. The specific enzyme activity was from 1 ,000 to 3,000 U/ml of the cultivation medium, from which the production strain biomass was separated. The volume obtainable by such method was in the range from 700 to 900 ml, i.e. the total yield of the obtained enzyme was from 700,000 U to 2,700,000 U of the human recombinant enterokinase, recalculated according to commercial standard EKMax™ (ThermoFisher Scientific). Figure 3: The enzyme profile of the human recombinant enterokinase in the medium during 7-day cultivation (168 hours). First line: molecule weights standard; second line: undiluted cultivation medium after 24 hours of the cultivation; third line: undiluted cultivation medium after 48 hours of the cultivation; fourth line: undiluted cultivation medium after 72 hours of the cultivation; fifth line: 1 ,000-times diluted cultivation medium after 96 hours of the cultivation: sixth line: 1 ,000-times diluted cultivation medium after 120 hours of the cultivation: seventh line: 1 ,000-times diluted cultivation medium after 144 hours of the cultivation: eighth line: 1 ,000-times diluted cultivation medium after 168 hours of the cultivation.

Example 3:

Controlled "fed-batch" cultivation of Pichia pastoris producer of the human recombinant enterokinase (rhEK)

Pichia pastoris culture prepared according to Example 1 was seeded on the fresh YPD (1 % yeast autolysate; 2% peptone; 2% glucose) agar plates, and cultivated at temperature of 29 °C. Afterwards one isolated colony from YPD plate was seeded to 50 ml of the inoculation medium (13.8 g/l (NH ₄ ) ₂ S0 ₄ , 46 g/l (NH ₄ )H ₂ P0 ₄ , 15.9 g/l KOH, 10 g/l glucose; 0.4 ml/l (v/v) 1 M MgS0 ₄ , 0.02 ml/l (v/v) 1 M CaCI ₂ , 4.6 ml/l PTM1 , 10g/I yeast autolysate, 20 g/l peptone) in 250ml Erienmeyer flask and the culture was cultivated 24 hours at temperature of 29 °C. The composition of microelements and growth substances solution PTM1 : 0.5 g/l CoCI ₂ .6H ₂ 0, 65 g/l FeS0 ₄ .7H ₂ 0, 3 g/l MnS0 ₄ .5H ₂ 0, 5 ml/l H ₂ S0 ₄ (95-98%), 0.08 g/l Kl, 6 g/l CuS0 ₄ .5H ₂ 0, 20 g/l ZnCI ₂ , 0.02 g/l H ₃ B0 ₃ , 0.2 g/l Na ₂ Mo0 ₄ .2H ₂ 0, and 0.2 g biotin.

The batch fermentation was carried out in the laboratory fermenter in total volume of 2 liters, at temperature of 29 °C, while 1000 ml of the following medium was used for the fermentation: 26.7 ml/l H ₃ P0 ₄ , 5.6 ml/l H ₂ S0 ₄ , 15.9 g/l (w/v) KOH, 10 g/l yeast autolysate and 20 g/l peptone. After the sterilization the medium was supplemented with 4.6 ml/l PMT1. The batch medium consisted of 500 g/l glucose, 100 mM MgS0 ₄ , 5mM CaS0 ₄ , and 12 ml/l PMT1. The cultivation conditions were controlled and maintained; particularly pH value of 6.0 via 30% solution of ammonium hydroxide, partial dissolved oxygen tension in medium (DOT) was kept at 60 % of its saturation value via automatic increasing of the agitator revolutions, as well as via manual adaptation of the volume in the air hoses for gases supplying into the cultivation media per time unit (vvm = volume of aeration per volume of medium per 1 minute), as well as via manual dispensing of the supplementing medium comprising carbon substrate, while the dispensing rate corresponded with the specific production strain biomass grow rate in the medium.

Upon obtaining of all desired parameters, at the beginning of the cultivation 30 ml of the feeding medium was supplied to the fermenter and 50 ml of inoculum from 24-hour cultivation by the pressing from the inoculation flask via air pressure by the Pichia pastoris strain carrying said construct with the gene encoding the human enterokinase light chain, in its chromosome, was inoculated to the fermenter. After the consumption of the carbon source contained in the basic cultivation medium the continual supplement feeding was initiated, while the feeding rate was exponential and adapted to the specific growth rate of the production strain culture at the level of 0.15 r ¹ . Such feeding mode was kept from hour 30 to hour 40 of the cultivation. With the increasing need of oxygen consumption the mixing revolutions were increasing automatically, and aeration was increasing manually. The aeration was increasing by such a way that at 500 rpm the aeration was set to 2 vvm, at 1000 rpm to 3 vvm, and at 1500 rpm to 4 vvm. After reaching the phase, when the production strain culture biomass had achieved the maximal rate of the carbon substrate utilization and this utilization rate had started to decrease gradually, at the keeping of the original feeding rate the biomass started to be oversaturated with the carbon substrate at such feeding rate. In that time the second phase of the cultivation started. Oversatu ration of the culture with the carbon substrate can be tested by a short stopping of the feeding. If the cells reacted by the reduced oxygen consumption sufficiently quickly within 30 seconds, it was a sign that the feeding rate is still acceptable for the cells.

In this phase of the cultivation the supplement feeding with the carbon substrate was decreasing by such a way to reduce the specific growth rate under 0.01 r ¹ , while the biomass growth was rather slower in this phase, in comparison with the first cultivation phase. The second cultivation phase lasted further 70 to 80 hours. In this phase the considerable accumulation of the human recombinant enterokinase in the medium occurred. The enzyme specific activity achieved the values from 900 to 2,500 U/ml of the cultivation medium filtrate. The volume obtainable by such method was in the range from 850 to 1 ,000 ml, i.e. the total yield of rhEK was from 765,000 U to 2,500,00U, resulting in the total yield of the obtained enzyme from 700,000 U to 2,700,000 U of ehEK, recalculated according to commercial standard EKMax™ (ThermoFisher Scientific).

Example 4:

Cultivation of P. pastoris in the laboratory fermenter for the production of rhEKL without initial batch phase.

P. pastoris culture prepared according to Example 1 was seeded on fresh YPD (1 % yeast autolysate; 2% peptone; 2% glucose) agar plates, and cultivated at temperature of 29°C. Afterwards one isolated colony from YPD plate was seeded to 50 ml of the inoculation medium (13.8 g/l (NH ₄ ) ₂ S0 ₄ , 46 g/l (NH ₄ )H ₂ P0 ₄ , 15.9 g/l KOH, 10 g/l glucose; 0.4 ml/l (v/v) 1 M MgS0 ₄ , 0.02 ml/l (v/v) 1 M CaCI ₂ , 4.6 ml/l PTM1 , 10g/I yeast autolysate, 20 g/l peptone) in 250ml Erlenmeyer flask and the culture was cultivated 24 hours at temperature of 29 °C. Solution of microelements and growth substances, PTM1 : 0.5 g/l C0CI2.6H2O, 65 g/l FeS0 ₄ .7H ₂ 0, 3 g/l MnS0 ₄ .5H ₂ 0, 5 ml/l H ₂ S0 ₄ (95-98%), 0.08 g/l Kl, 6 g/l CuS0 ₄ .5H ₂ 0, 20 g/l ZnCI ₂ , 0.02 g/l H3BO3, 0.2 g/l Na ₂ Mo0 ₄ .2H ₂ 0, and 0.2 g biotin. The supplement feeding fermentation was carried out in the laboratory fermenter in total volume of 2 liters, at temperature of 29 °C, while 750 ml of the following medium was used for the fermentation: 26.7 ml/l H ₃ P0 ₄ , 5.6 ml/l H ₂ S0 ₄ , 15.9 g/l (w/v) KOH , 10 g/l yeast autolysate and 20 g/l peptone. After the sterilization the medium was supplemented with 4.6 ml/l PMT1. The supplement medium consisted of 500 g/l glucose, 100 mM MgS0 ₄ , 5mM CaS0 ₄ , and 12 ml/l PMT1. The cultivation conditions were controlled and maintained; particularly pH 6.0 via 30% ammonium hydroxide, DOT was kept at 60 % of its saturation value via automatic increasing of the agitator revolutions, further via manual adaptation of the volume in the air hoses for gases supplying into the cultivation media per time unit (vvm = volume of aeration per volume of medium per 1 minute), as well as via manual supply of the supplement medium according to the specific biomass grow rate in the medium.

Upon obtaining of all desired parameters the constant continual feeding of the supplement medium at 0.1 ml/min. was initiated from the beginning of the cultivation and the fermenter was inoculated with 50 ml of an inoculum from 24-hour cultivation of P. pastoris strain carrying the above construct with the gene encoding the human enterokinase light chain in its chromosome. At the beginning of the cultivation the cells grew with the maximal specific growth rate and the carbon substrate was partially accumulated in the medium, but without obtaining as high concentration as at the beginning of the cultivation in the case of "batch" cultivation, being about 20 g/l of glucose. By this process the inhibition effect of too high concentration of the substrate was eliminated. Upon obtaining of a biomass concentration having capacity to consume the preset supplement feeding, the feeding of the supplement medium was set identically as in Example 2. It means that the supplement feeding rate was adapted to the biomass growth rate. Feeding rate had been increasing until the cells reacted to the increasing by the increasing of oxygen consumption. Upon obtaining of a supplement feeding rate, at which the cells stopped to react, the supplement feeding rate was reduced and set to a value, at which the culture still reacted. With the increasing need of oxygen consumption by the biomass the mixing revolutions were increasing automatically and aeration was increasing manually. The aeration value was increasing at 500 rpm to 2 vvm, at 1000 rpm to 3 vvm, and at 1500 rpm to 4 vvm. By this method the specific growth rate was kept above 0.05 r ¹ until obtaining the revolutions of 2,000 rpm, and the aeration of 4 vvm. Such feeding mode was kept during 40 to 60 hours of the cultivation.

In this phase of the cultivation the supplement feeding was decreasing by such a way to reduce the specific growth rate under 0.01 hr ¹ , while the biomass growth was rather slower in this phase, in comparison with the first cultivation phase. The second cultivation phase lasted from hour 70 to hour 80. During this phase the considerable accumulation of the human recombinant enterokinase in the medium occurred. The specific enzyme activity achieved the values from 1 ,000 to 3,000 U/ml of the cultivation medium filtrate. The volume obtainable by such method was in the range from 700 to 900 ml, i.e. the total yield was from 700,000 U to 2,700,000 U of rhEK, which is the activity in international units recalculated according to commercial standard EKMax™ (ThermoFisher Scientific).

The human recombinant enterokinase (rhEK) was identified in the medium and isolated using an affinity chromatography. The isolated enzyme was applied on SDS PAGE electrophoresis and identified as a blur in the range from 70 to 130 kDa due to non- homogenous enterokinase glycosylation. After deglycosylation, e.g. by EndoHF, the strict band was identified at the level of the desired molecular weight (26.9 kDa). Further the enzyme was tested for enzyme kinetics on fluorogenic substrate comprising the target amino acids sequence (GD4K-na), where the enzyme achieved Km and K _ca t value comparable with those published in the works relating to the production of the recombinant human enterokinase. Furthermore, a C-terminal sequence of 6 histidines was identified by Western blotting. By the above methods the identity of the human enterokinase light chain enzyme was confirmed.

A method for cultivation according to this invention describes processes, methods, and product yields enabling the preparation of the commercial amounts of the human recombinant enterokinase enzyme. Sequence Listing

<110> Jan Krahulec, Department of Molecular Biology, Prif UK Bratislava

<120> Method of cultivation of Pichia pastoris for production of human enterokinase

<160> 3

<210> 1

<211> 483

<212> DNA

<213> Pichia pastoris

<400>

aga tct ttt ttg tag aaa tgt ctt ggt gtc etc gtc caa tea ggt age cat 51 etc tga aat ate tgg etc cgt tgc aac tec gaa cga cct get ggc aac gta 102 aaa ttc tec ggg gta aaa ctt aaa tgt gga gta atg gaa cca gaa acg tct 153 ctt ccc ttc tct etc ctt cca ccg ccc gtt acc gtc cct agg aaa ttt tac 204 tct get gga gag ctt ctt eta egg ccc cct tgc age aat get ctt ccc age 255 att acg ttg egg gta aaa egg agg teg tgt acc cga cct age age cca ggg 306 atg gaa aag tec egg ccg teg ctg gca ata ata gcg ggc gga cgc atg tea 357 tga gat tat tgg aaa cca cca gaa teg aat ata aaa ggc gaa cac ctt tec 408 caa ttt tgg ttt etc ctg acc caa aga ctt taa att taa ttt att tgt ccc 459 tat ttc aat caa ttg aac aac tat 483

<210> 2

<211> 276

<212> DNA

<213> Saccharomyces cerevisiae

<400>

1 ttc gaa acg atg agd tty ccw tch aty ttc ach gch gty ttr ttt gch gch 51

Met Arg Phe Pro Ser lie Phe Thr Ala Val Leu Phe Ala Ala

1 5 10

tch tch gch ttr gch gch ccw gty aay ach ach ach gar gay gar acn gch 102 Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala

15 20 25 30

car aty ccn gch gar gch gty aty ggw tay tch gay ttr gar ggn gay tty 153 Gin lie Pro Ala Glu Ala Val lie Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 50 55

gay gty gch gty ttr ccw tty tch aay agh ach aay aay ggn ttr ttr tty 204 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe

60 65 70 ath aay ach ach aty gch age aty gch gch aar gar gar ggn gtn tch ctn 255 He Asn Thr Thr He Ala Ser He Ala Ala Lys Glu Glu Gly Val Ser Leu

75 80 85 90 gar aar agd gar gch gar gch

Glu Lys Arg Glu Ala Glu Ala

95

<210> 3

<211> 723

<212> DNA

<213> Homo sapiens

<400>

1 aty gty ggw ggw tcy aay gch aar gar ggw gch tgg ccw tgg gty gty ggw 51

He Val Gly Gly Ser Asn Ala Lys Glu Gly Ala Trp Pro Trp Val Val Gly

1 5 10 15

ttd tay tay ggw ggw agd ttd ttd tgy ggw gch tcy ttd gty tcy tcy gay 102

Leu Tyr Tyr Gly Gly Arg Leu Leu Cys Gly Ala Ser Leu Val Ser Ser Asp

20 25 30

tgg ttd gty tcy gch gch cay tgy gty tay ggw agd aay ttd gar ccw tcy 153

Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Leu Glu Pro Ser 35 40 45 50 aar tgg ach gch aty ttd ggw ttd cay atg aar tcy aay ttd ach tcy ccw 204

Lys Trp Thr Ala He Leu Gly Leu His Met Lys Ser Asn Leu Thr Ser Pro

55 60 55 car ach gty ccw agd ttd aty gay gar aty gty aty aay ccw cay tay aay 255 Gin Thr Val Pro Arg Leu He Asp Glu He Val He Asn Pro His Tyr Asn

70 75 80 85 agd agd agd aar gay aay gay aty gch atg atg cay ttd gar tty aar gty 306 Arg Arg Arg Lys Asp Asn Asp He Ala Met Met His Leu Glu Phe Lys Val

90 95 100

aay tay ach gay tay aty car ccw aty tgy ttd ccw gar gar aay car gty 357 Asn Tyr Thr Asp Tyr He Gin Pro He Cys Leu Pro Glu Glu Asn Gin Val

105 110 115

tty ccw ccw ggw agd aay tgy tcy aty gch ggw tgg ggw ach gty gty tay 408 Phe Pro Pro Gly Arg Asn Cys Ser He Ala Gly Trp Gly Thr Val Val Tyr 120 125 130 135 car ggw ach ach gch aay aty ttd car gar gch gay gty ccw ttd ttd tcy 459 Gin Gly Thr Thr Ala Asn He Leu Gin Glu Ala Asp Val Pro Leu Leu Ser 140 145 150

aay gar agd tgy car car car atg ccw gar tay aay aty ach gar aay atg 510 Asn Glu Arg Cys Gin Gin Gin Met Pro Glu Tyr Asn lie Thr Glu Asn Met

155 160 165 170 aty tgy gch ggw tay gar gar ggw ggw aty gay tcy tgy car ggw gay tcy 561 lie Cys Ala Gly Tyr Glu Glu Gly Gly lie Asp Ser Cys Gin Gly Asp Ser

175 180 185

ggw ggw ccw ttd atg tgy car gar aay aay agd tgg tty ttd gch ggw gty 612 Gly Gly Pro Leu Met Cys Gin Glu Asn Asn Arg Trp Phe Leu Ala Gly Val

190 195 200

ach tcy tty ggw tay aar tgy gch ttd ccw aay agd ccw ggw gty tay gch 663 Thr Ser Phe Gly Tyr Lys Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala

205 210 215 220 agd gty tcy agd tty ach gar tgg aty car tcy tty ttd cay cay cay cay 714 Arg Val Ser Arg Phe Thr Glu Trp lie Gin Ser Phe Leu His His His His

225 230 235

cay cay tag

His His ***

240