SK500482016A3 - DNA expression cassette for human entherokinase light chain gene, stem Pichia pastoris comprising the DNA expression cassette and method of its cultivation - Google Patents

Abstract

The expression DNA cassette consists of at least a yeast promoter region, followed by a functionally linked nucleotide sequence encoding a secretion signal together with a nucleotide sequence that encodes a site for appropriate proteolytic processing to cleave the secretory signal. Further, the expression DNA cassette after the secretion signal carries a functionally bound nucleotide sequence encoding the human enterokinase light chain with 6 histidine at the C-terminus of the enterokinase gene. Said expression DNA cassette is integrated in the genome of Pichia pastoris. Also described is a method for culturing said human recombinant enterokinase producing Pichia pastoris. The method of cultivation consists in biphasic cultivation in a fermenter, wherein in the first phase the cells are cultured at a high specific biomass growth rate, where a significant increase in biomass occurs at this stage and no enterokinase accumulation occurs in the culture medium. This phase is followed by the second production phase of the culture, which is carried out at a low specific growth rate of the biomass cells, resulting in minimal growth of biomass, but significant production and accumulation of enterokinase occurs in the culture medium.

Description

Specific activity (U / ml)

An expression DNA cassette carrying a gene encoding a human enterokinase light chain, a Pichia pastoris strain carrying said expression DNA cassette, and a method for culturing it.

Technical field

The invention relates to a yeast Pichia pastoris carrying an integrated expression DNA cassette in its genome, to an expression DNA cassette and to a method of culturing the yeast Pichia pastoris for the production of human recombinant enterokinase in the form of a soluble protein.

BACKGROUND OF THE INVENTION

Enterokinase (EC 3.4.21.9) is a serine protease of the small intestinal mucosa mucosa. Physiologically, enterokinase activates the native trypsinogen substrate to trypsin by cleavage of the N-terminal portion of the peptide, followed by the conserved sequence of four aspartic acids and one lysine (Asp) 4-Lys (Li, 1997). It is this sequence of five amino acids that is highly specifically recognized by enteropeptidase, which cleaves the N-terminal protein from the C-terminal just after this amino acid sequence. Cleavage of the N-terminal portion produces active trypsin, which in turn activates many other pancreatic zymogens (Kunitz, 1939). The native enteropeptidase dimer was isolated from porcine (Matsushima et al., 1994), bovine (Kitamoto, 1994), mouse (Yuan et al., 1998), rat (Yahagi, 1996), Japanese rice fish (Ogiwara and Takahashi , 2007) and the human (Kitamoto et al., 1995) intestines. In all cases, the enteropeptidase appears to be a heterodimer whose chains are linked by a disulfide bond. The enteropeptidase precursor is a single-stranded polypeptide composed of a heavy (82-140 kDa) and a light (35-62 kDa) chain. The heavy chain containing the N-terminal membrane domain has little effect on the recognition of cleavage sites in small peptides, but has a strong effect on substrate recognition when it is a macromolecular substrate and also has a large effect on specificity

-2 inhibitor (Lu et al., 1997). The light chain is a catalytic subunit of enteropeptidase and contains a serine protease-like domain of chymotrypsin (Lu and Sadler, 1998). The high degree of specificity that enterokinase has makes this enzyme ideal for cleaving affinity tags or fusion proteins, thereby distinguishing them from the target recombinant protein produced in bacteria. Expression and purification of the recombinant bovine enteropeptidase catalytic unit has been described for various host strains such as Escherichia coli (Collins-Racie et al., 1995; Yuan and Hua, 2002; Tan et al., 2007), yeast Saccharomyces cerevisiae (Choi et al. , 2001), methylotrophic yeast Pichia pastoris (Vozza et al., 1996), filamentous fungus Aspergillus niger (Svetina et al., 2000) and for monkey kidney COS-1 cells (Vallie et al., 1993).

In addition, human enteropeptidase was also expressed in E. coli cells. Peptidase was expressed in fusion with thioredoxin as inclusion bodies and was renatured by the dilution method. By dilution dilution only a maximum of 2% of the active peptidase could be obtained, but the activity of its active moieties was approximately 5-fold higher than that of recombinant bovine enteropeptidase derived from P. pastoris yeast. This is also confirmed by the measured K _m and k cat values of the constants of this enzyme (Gasparian 'et al., 2003). The results of enterokinase expression in E. coli cells published by various authors are considerably inconsistent. While Gasparian (2003) et al. have been able to refold human enterokinase in approximately 2% of inclusion bodies, although more than 90% of the inclusion body product was at 37 ° C, Liu and Hua (2002) expressed bovine enzyme in the same vector as soluble and active with a specificity constant approximately equal to that measured by Gasparian et al. (2003) for bovine enteropeptidase expressed in P. pastotris. It should be noted that Gasparian et al. (2003) showed no activity in the soluble fraction. Enteropeptidase was also expressed in fusion with glutathione S-transferase, where 90% of the product was formed as inclusion bodies. The total amount of mature enterokinase obtained after purification, refolding and autocatalytic digestion of the enzyme was 27.5 mg per 1 L of fermentation culture. The activity of pure enteropeptidase was comparable to that of commercial enterokinase provided by Sigma (Tan et al., 2007).

-3Bovine enterokinase in P. pastoris cells was first produced in

1996 (Vozza et al., 1996). After purification, a yield of pure peptidase of 6.3 mg.l ^{-1 of} fermentation medium was obtained, with a specific activity of 168 µm ^-1 , which was more than was achieved when expressed in E. coli (Collins-Racie et al., 1995).

In 2004 (Fang et al.), An attempt was made to produce bovine enterokinase in P. pastoris cells under the control of the pAOX promoter. The amount of secreted bovine enterokinase in the medium was 10 mg.l ^{-1 of the} fermentation culture. To improve the purification of the enzyme in the following downstream step, a His-tag was attached to the DNA sequence encoding the enterokinase. The yield of enterokinase reached 5.4 mg.l ^{-1 of a} fermentation medium with an activity of 80 U.mg ^-1 (Fang et al., 2004), which is an even lower yield than that reported in the previous publication (Vozza et al.1996). The bovine enteropeptidase thus obtained was able to cleave almost 100% of the substrate in 16 hours at 16 ° C (Fang et al., 2004), when the ratio of the substrate, the GSTvazostatin fusion protein, including the bEK cleavage site, to the enzyme enterokinase 1000: 1. The following research focused on increasing the yield of bovine recombinant enterokinase by monitoring the degree of strain selection (methanol utilizing the host phenotype), further optimizing the fermentation conditions in terms of the pH effect during cultivation as well as the carbon source used on the expression level. Using strains with methanol-induced promoters under the control of the pAOX promoter, the MutS strain proved to be more suitable for the production of recombinant bovine enterokinase than the Mut + strain. The advantage of the MutS strain is its reduced sensitivity to residual methanol in the culture medium as opposed to the Mut + strain. After 120 hours induction in medium with glycerol and methanol, at pH 6, an expression rate with an enterokinase production of 350 mg.l ^-1 fermentation medium was achieved. After purification, 150 mg of pure enzyme per liter of medium were obtained, with a specific activity of 9000 U (International Units of Enzyme Activity) .mu.g ^-1 (Peng et al., 2004). This was twice as low as the enterokinase produced by the filamentous fungus A. niger (Svetina et al., 2000). When the His-tag was attached to the C-terminus of the gene encoding bovine enterokinase, the specific enzyme activity was reduced to 8000 U. mg ^-1 (Peng et al., 2004).

The rate of heterologous protein production depends not only on the conditions and parameters of the culture process but also on the selection of the promoter to which Pepeliaev et al., (2011). In some cases, constitutive expression of recombinant proteins with a pGAP promoter achieves higher production than that of classical expression under the control of an inducible pAOX promoter. The use of a pGAP promoter design presents certain advantages. One is the elimination of methanol as an inducer and carbon source during fed-batch culture, thereby reducing cell lysis and consequently the proteolytic activity of secreted proteases (Zhang et al., 2009). By comparing the expression rate of human enterokinase using different promoters, the highest expression rate was observed in the experiments using the pGAP promoter. After 120 hours of culture, an enterokinase concentration of 73.5 mg.l ^{-1 of} culture medium with an activity of 20,000 U.l ^-1 was observed. The production rate was calculated based on the specific activity of the commercial bovine enterokinase EKMax® (Invitrogen), which showed three times lower activity than human recombinant enterokinase.

The production of enteropeptidase in the Aspergillus niger expression system was discussed in Svetina et al. (2000), which expressed the cDNA encoding the catalytic subunit of bovine enterokinase in fusion with the linker protein glucoamylase 2. After purification by ion-exchange chromatography, they yielded a 1 mg.l- ¹ culture medium of the highly active enteropeptidase enzyme which reached 10 times greater than ¹ compared to commercial EKMax® (Invitrogen) (Svetina et al., 2000).

US 2015/0020238 A1 discloses a plant cell transformed with a recombinant vector comprising a synthetic gene encoding a human enterokinase light chain. Rice is used as a plant cell.

WO2012071257 (US 8,557,558) describes the preparation of polynucleotide molecules encoding bovine enterokinase, further yeast expression constructs comprising a yeast expression vector, and polynucleotide molecules encoding a bovine enterokinase, yeast cells containing the expression entero construct, or a cleavage method, or a cleavage method. .

-5-Summary of the invention

The present invention provides an expression DNA cassette carrying a gene encoding the human enterokinase light chain of SEQ ID NO: 3.

According to another embodiment, the present invention provides an expression DNA cassette which carries a gene encoding the secretory signal α-Mating Factor of Saccharomyces cerevisiae according to SEQ ID NO: 2, which is fused to the 5'-end of the human enterokinase light chain gene according to SEQ ID NO : 3 in the same direction.

Another object of the invention is an expression DNA cassette which carries a promoter from the glyceraldehyde phosphate dehydrogenase gene of SEQ ID NO: 1 operably upstream of the gene encoding the aMF secretion signal of Saccharomyces cerevisiae according to SEQ ID NO: 2 which is fused to the 5'- the end of the gene encoding the human enterokinase light chain according to SEQ ID NO: 3 in the same direction.

According to a preferred embodiment of the invention, the expression DNA cassette carries a promoter from the glyceraldehyde phosphate dehydrogenase gene according to SEQ ID NO: 1 operably upstream of the gene encoding the aMF secretion signal from Saccharomyces cerevisiae according to SEQ ID NO: 2 which is fused to the 5 'end of the gene encoding a human enterokinase light chain according to SEQ ID NO: 3 in the same direction,. wherein the sequences encoding proteolytic cleavage sites for Kex2 and Ste13 protease genes are located between the secretion signal coding sequence and the gene encoding the human enterokinase light chain of SEQ ID NO: 3, wherein the human enterokinase light chain after proteolytic cleavage at its N-terminus contains only amino acid human enterokinase light chain residues.

The present invention also provides a high-production strain of Pichia pastoris, carrying the above-described expression DNA cassette, which is integrated in its chromosome.

The present invention also provides a method of culturing a high-production strain of the Pichia pastoris strain carrying the above-described expression DNA cassette integrated in its chromosome for the production of human enterokinase light chain. The cultivation process is conducted in two phases, wherein the first growth phase sets the culture conditions to maintain a high specific biomass growth rate of at least 0.02 h ^-1 and the second production phase of the culture sets the culture conditions to maintain a low specific biomass growth rate , which is at most 0.01 h ^-1 , minimizing biomass growth at this stage of cultivation, but significantly producing and accumulating recombinant human enterokinase in the culture medium as a soluble protein.

According to a preferred embodiment, the first growth phase of the cultivation runs from the beginning of the cultivation up to the 72nd hour of cultivation, wherein the exponential increase in biomass of the production strain expressed as biomass dry weight is recorded and then conditions for the second production stage of cultivation of the production strain are set within 24 to 36 hours after 72 hours of culture, wherein the second production phase lasts 60 to 80 hours during which recombinant human enterokinase accumulates in the culture medium in the form of a soluble protein.

According to a further preferred embodiment, the process control conditions for aeration and mixing are set in the first growth phase to achieve a high dissolved oxygen partial pressure (DOT) of 55-65% saturation and a controlled specific feed of the biomass with the carbon substrate to provide a high specificity. biomass growth rate, expressed as biomass dry weight (DCW), in the range of 150-200 g / L culture medium.

The subject of the invention is an expression DNA cassette comprising at least a yeast promoter region, for example a glyceraldehyde phosphate dehydrogenase gene promoter, after which a nucleotide sequence encoding a

A secretion signal together with a nucleotide sequence that encodes a site suitable for proteolytic processing to cleave the secretion signal. Further, the expression DNA cassette carries a functionally linked nucleotide sequence encoding a light chain of human enterokinase with 6 histidines inserted at the C-terminus of the enterokinase gene downstream of the secretion signal. The sequences included to separate the secretion signal from the human enterokinase light chain coding sequence are situated in such a way that no amino acid residues not belonging to the human enterokinase light chain sequence remain at the N-terminus of the human enterokinase gene after processing.

The subject of the invention is also the yeast Pichia pastoris, which carries in its genome an integrated described expression DNA cassette, in one or more copies.

The present invention also provides a method for culturing a modified yeast Pichia pastoris which carries genetic information encoding a human enterokinase light chain integrated in a chromosome. This genetic information is operably linked to a constitutive promoter allowing expression of said genetic information into an active enzyme form. Included in the genetic information is a secretory signal that allows the enzyme to be expressed into the extracellular space, with a proteolytic cleavage site between the secretory signal and the human enterokinase light chain to ensure removal of the secretory signal during the secretory pathway without leaving amino acid residues at the N-terminus of the polynucleotide encoding enterokinase . Also included in the genetic information is a sequence encoding the 6 histidines at the C-terminus of the polynucleotide to facilitate purification of the enzyme.

Using the method of culturing the genetically engineered Pichia pastoris yeast of the present invention, there is a significant increase in recombinant human enterokinase production over the available prior art information. The method of cultivation consists of two-phase cultivation.

In both phases of cultivation, a complex feed containing glucose as a carbon source is continuously added to the culture medium, the feed rate being adapted to the growth rate of the culture and to the technical specification of the parameters of the fermenter.

An important parameter for controlling the culture process is to maintain the Dissolved Oxygen Tension (DOT) partial pressure above 30% saturation, since the physiological processes in P. pastoris yeast are most effective in aerobic mode. In the first growth phase of the culture, the DOT level is maintained by increasing mixing and aerating the medium. Such regulation can be achieved either manually or if the fermentation plant is equipped with an automatic system for increasing the speed depending on the oxygen consumption, this process can be conducted semi-automatically with manual increase in aeration, i.e. continuous air supply below the culture medium. If the system is equipped with a multi-level automatic control system not only for mixing speed, but also for automatic aeration control (Gass Flow Controller) and gas mixing (Gass Mix), this process can be fully automated. With an automatic gas mixer, the air mixture can be enriched with oxygen and thereby increase not only the efficiency of the first phase of biomass growth, but also the second phase of human enterokinase production.

Throughout this first phase of cultivation, the feeding process, i.e. the feeding of the carbon substrate in solution, is conducted so that the feeding rate provides a specific biomass growth rate that is just below the maximum specific growth rate. The feed rate is controlled by the biomass reaction to oxygen consumption. If the cells stop responding with increased oxygen consumption as the dosage increases, the dosage is greater than or equal to that which provides the maximum specific growth rate. The specific growth rate during the first growth phase of the culture should not fall below 0.02 h ^-1 , preferably below 0.05 h ^-1 . The first cultivation phase ends either by reaching specific parameters of the fermenter or by reaching a biomass density such that the specific growth rate begins to fall sharply and fall to or below 0.01 h ^-1 . The second production phase of cultivation is characterized by a low specific growth rate ranging from 0.01 h ^-1 to 0.001 h ^-1 , while reducing the feeding rate so as to maintain the maximum agitation parameters achieved throughout the second phase of cultivation, aeration and addition of a mixture of gases, i.e. air and oxygen. During this phase, the rate of oxygen consumption increases to a given feed rate. In other words, at a constant speed

For example, in order to maintain the desired DOT, it is necessary to reduce the feed rate of the feed in order to maintain the desired DOT. During this second production phase of culture, there is a significant accumulation of enterokinase in the medium.

The high cell density of the Pichia pastoris production strain (High Cell Density Cultivation) allows the cultivation period to be extended to 140-160 hours. In this second phase, there is a significant increase in recombinant human enterokinase (rhEK) production in the range of 1000 to 3000 U / ml culture medium filtrate. This recombinant human enterokinase is excreted from the cells of the Pichia pastoris production strain into the medium as a soluble protein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a record of electrophoretic testing of the expression ability of individual clones tested by bank culture. Figure 2 is a graphical representation of the cultivation profile of P. pastoris strain in two-phase culture fermentation in a laboratory fermentor. In Figure 3, the enzyme profile of enterokinase activity in the medium is tested during a 7 day culture (168 hours).

DETAILED DESCRIPTION OF THE INVENTION

Example 1: Preparation of a production strain to produce rhEKL.

The plasmid pGZE with the zeocin resistance gene and the promoter from the glyceraldehyde phosphate dehydrogenase (GAP) gene from Pichia pastoris was used to transform electro-competent cells of the wild Pichia pastoris Y11430 strain followed by a functionally coding for the alpha-mating factor polypeptide gene. (and MF), both as a secretion signal and the human enterokinase light chain gene with his-tag amino acid motif at the C-terminus (HLEKh). Between aMF and HLEKh are further

It finds a Kex2 protease cleavage site and target sequences for the Ste13 gene encoding a dipeptidyl aminopeptidase so that the human enterokinase light chain after proteolytic processing at its N-terminus does not contain any non-human enterokinase light chain amino acid residues. The plasmid was linearized by restriction endonuclease AvrII prior to electroporation and purified using a DNA fragment purification kit. An aliquot of 50 µl of electro-competent cells was transformed with a linearized plasmid at a total of 5 µg and the transformed cells were selected on solid YPD (1% yeast autolysate; 2% peptone; 2% glucose) with zeocin addition (100 µg / ml). Transformants were cultured in shaker flasks under selection conditions at 29 ° C for 3 days. Approximately 20 colonies were selected from this medium as positive transforms. These selected colonies were inoculated into 2 ml of YPD liquid culture medium with the addition of zeocin and cultured at 29 ° C for 24 hours on a shaker.

After cultivation, chromosomal DNA was isolated from the selected clones, and the presence of an integrated expression plasmid was detected by PCR. The presence of the expression plasmid was detectable in almost all clones. Clones that were found to have plasmid DNA in the chromosome were tested for the presence of human enterokinase enzyme activity in the culture medium without the addition of an antibiotic. From 2 ml of Pichia pastoris night culture from selected clones in YPD medium, 20 μΙ was inoculated into 20 ml of new YPD medium and cultured for 144 hours with 1 ml of 50% sterile glucose solution added to the culture each day. After the culture was complete, the cells were separated from the medium by centrifugation. The human enterokinase activity assay was performed by applying the obtained clone assay medium to a 20 µg protein substrate carrying an enterokinase cleavage site in a volume of 5 μΙ in 10 mM TrisHCl, pH8. The medium was diluted 10 and 100 times (Fig. 1). The cleavage efficiency was evaluated by the GeneTools densitometric software from SynGene. The best efficiency clone was selected to further test enterokinase production by fermentation in larger volumes.

Figure 1 shows a record of electrophoretic testing of the productivity of individual clones in bank expression. Lane 1: molecular weight standard;

lane 2: uncleaved substrate; 3: substrate cleaved by commercial enterokinase; lanes 410: 10x diluted media clones; lanes 11-17: respective clones with 100x diluted media.

Example 2: Cultivation of Pichia pastoris in a laboratory fermentor to produce rhEKL by an initial batch process.

Selected clones of Example 1 were inoculated onto fresh YPD (1% yeast autolysate; 2% peptone; 2% glucose) agar plates and cultured at 29 ° C statically. Then, one isolated YPD colony was inoculated into 50 ml of inoculum medium (13.8 g / l (N1-kSCU, 46 g / l (NH4) H2PO4, 15.9 g / l KOH, 10 g / l glucose; 0.4 ml / l ( v / v) 1M MgSO4, 0.02 ml / l (v / v) 1M CaCl2, 4.6 ml / l PTM1, 10g / l yeast autolysate, 20 g / l peptone) in a 250 ml Erlenmayer flask and cultured for 24 hours at temperature 29 ° C. A solution of PTM1-labeled microelements and growth agents was added to the medium with the following composition: 0.5 g / l COCl 2 .6H2, 65 g / l FeSO4.7H2O, 3 g / l MnSO4.5H2O, 5 ml / l H2SO4 ( 95-98%), 0.08 g / l K1, 6 g / l CuSO4.5H2O, 20 g / l ZnCl2, 0.02 g / l H3BO3, 0.2 g / l Na2MoO4.2H2O and 0.2 g biotin.

The supplementary fermentation was carried out in a laboratory fermentor with a total volume of 2 liters at 29 ° C, using 750 ml of medium for cultivation: 26.7 ml / l H 3 PO 4, 5.6 ml / l H 2 SO 4, 15.9 g / l (w / v) KOH, 10 g / l yeast autolysate and 20 g / l peptone. After sterilization, the medium was supplemented with 4.6 ml / L PMT1. The feed medium was composed of 500 g / L glucose, 100 mM MgSO 4, 5 mM CaSO 4 and 12 mL / L PMT1. The culture conditions were controlled and maintained as follows: pH 6.0 with 30% ammonium hydroxide, dissolved oxygen partial pressure in the medium (DOT) was maintained at 60% saturation by automatically increasing the stirrer speed, and manually adjusting the volume of air gases fed to the culture medium per unit time (vvm = volume of

-12aeration per volume of medium per 1 minute), as well as by manually dosing the feed medium according to the specific biomass growth rate in the medium.

Upon reaching all the required parameters, 30 ml of feed medium was dosed into the fermenter at the beginning of the culture and the fermentor was inoculated with 50 ml of inoculum from a 24 hour culture using an inoculum bottle and air pressure, Pichia pastoris strain carrying a chromosome construct with human enterokinase light chain gene. is described in detail above. After the carbon source contained in the basic culture medium was consumed, continuous feeding of the feed solution containing the identical carbon substrate was started, the feeding rate being adapted to the cell growth rate. The dosing rate was increased until the cells reacted to increasing the concentration of the carbon substrate contained in the medium by increased oxygen consumption. At such a feed rate that the cells stopped responding to an increased feed and consequently an increased concentration of the carbon substrate in the medium, the feed rate decreased to the last value at which the culture was still reacting with increased dissolved oxygen consumption. With increasing oxygen demand, the stirring speed was automatically increased and the aeration was increased manually. The aeration was increased so that at 500 rpm (revolution per minute) the aeration was set at 2 wm, at 1000 rpm at 3 wm and at 1500 rpm at 4 wm. In this way, the specific biomass growth rate was maintained above 0.05 h ^-1 until 2000 rpm and aeration of 4 wm were reached. This phase lasted from 40 to 60 hours of culture.

After reaching such cultivation parameters (Fig. 2), the second phase of growth of the culture was followed, when the feed rate was reduced so that the specific growth rate fell below 0.01 h-1, while the biomass growth in this phase was already quite slow compared to the first phase (Fig. 2). The second phase of the culture lasted 70-80 hours. At this stage, there was considerable accumulation of human recombinant enterokinase (rhEK) in the medium as a soluble protein, with its highest concentrations measured at 144-168 hours of culture (Fig. 2, 3). The activity of rhEK in the fermented medium with the production strain Pichia pastoris was tested using a commercial substrate Trx-DCD1, which carries a site for specific cleavage by enterokinase. specific

The 13-enzyme activity was 1000 to 3000 U / ml culture medium from which the biomass of the production strain was separated. The volume of media obtainable in this manner ranged from 700 to 900 ml, which meant a total yield of the obtained enzyme of 700,000 U to 2,700,000 U of human recombinant enterokinase recalculated according to the commercial standard EKMax ™ (ThermoFisher Scientific).

Figure 3: Enzyme profile of human recombinant enterokinase activity in medium over 7 days of culture (168 hours). First path: molecular weight standard; second lane: undiluted culture medium after 24 hours of culture; third lane: undiluted culture medium after 48 hours of culture; fourth lane: undiluted culture medium after 72 hours of culture; fifth lane: 1000-fold diluted culture medium after 96 hours of culture; sixth lane: 1000-fold diluted culture medium after 120 hours of culture; seventh lane: 1000-fold diluted culture medium after 144 hours of culture; Eight lane: 1000-fold diluted culture medium after 168 hours of culture

Example 3:

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

A Pichia pastoris culture prepared according to Example 1 was inoculated onto fresh YPD (1% yeast autolysate; 2% peptone; 2% glucose) agar plates and cultured at 29 ° C. Then, one isolated YPD colony was inoculated into 50 ml of inoculation medium (13.8 g / l (NH ₄ ) ₂ SO ₄ , 46 g / l (NH ₄ ) H ₂ PO ₄ , 15.9 g / l KOH, 10 g / l glucose; 0.4 ml / l (v / v) 1M MgSO ₄ , 0.02 ml / l (v / v) 1M CaCl ₂ , 4.6 ml / l PTM1, 10g / l yeast autolysate, 20 g / l peptone) in 250 ml Erlenmayer flask and culture was cultured for 24 hours at 29 ° C. Composition of the solution of microelements and growth substances PTM1: 0.5 g / l CoCl ₂ .6H ₂ , 65 g / l FeSO ₄ .7H ₂ O, 3 g / l MnSO ₄ .5H ₂ O, 5 ml / l H ₂ SO ₄ (9598 %), 0.08 g / l Kl, 6 g / l CuSO ₄ .5H ₂ O, 20 g / l ZnCl ₂ , 0.02 g / l H ₃ BO3, 0.2 g / l Na ₂ MoO ₄ .2H ₂ O and 0.2 g biotin.

The 14-feed fermentation was carried out in a laboratory fermentor with a total volume of 2 liters at 29 ° C, using 1000 ml of medium for culture: 26.7 ml / l H 3 PO 4. 5.6 ml / l H2SO4, 15.9 g / l (w / v) KOH, 10 g / l yeast autolysate and 20 g / l peptone. After sterilization, the medium was supplemented with 4.6 ml / L PMT1. The feed medium was composed of 500 g / L glucose, 100 mM MgSO 4, 5 mM CaSO 4 and 12 mL / L PMT1. The culture conditions were controlled and maintained at pH 6.0 with 30% ammonium hydroxide solution, the dissolved oxygen partial pressure (DOT) was maintained at 60% saturation by automatically increasing the stirrer speed, as well as by manually adjusting the volume of air feed gases into the culture medium per unit of time (wm = volume of aeration per volume of medium per minute), and also by manual feeding of the feed medium containing the carbon substrate, the feed rate corresponding to the specific biomass growth rate of the production strain in the medium.

After all the desired parameters had been reached, 30 ml of feed medium was dosed into the fermenter at the beginning of the culture and the fermentor was inoculated with 50 ml of inoculum from a 24 hour culture by extrusion from an inoculum flask by air pressure of P. pastoris carrying the aforementioned human enterin light chain gene. After the carbon source contained in the basic culture medium was consumed, a continuous feed was started, the feed rate being exponential and adapted to the specific growth rate of the production strain culture at 0.15 h ^-1 . Such a dosing method was maintained from 30 to 40 hours of culture. As the oxygen demand of the culture increased, the stirring speed was automatically increased and the aeration manually increased. The aeration was increased so that at 500 rpm the aeration was set at 2 vvm, at 1000 rpm at 3 wm and at 1500 rpm at 4 wm. After reaching the phase where the biomass of the production strain culture reached the maximum utilization rate of the carbon substrate and this utilization rate began to gradually decrease, while maintaining the original feed rate, the biomass began to be presaturated with the carbon substrate at such feed rate. Then the second phase of cultivation began. Presaturation of the culture with a carbon substrate could be tested

-15 short-term dosing stoppages. If the cells responded quickly enough with reduced oxygen consumption within 30 seconds, this indicated that the dosing rate was still acceptable to the cells.

After this cultivation phase, the feed of the carbonaceous substrate was reduced so that the specific growth rate fell below 0.01 h -1, and the biomass growth in this phase was already considerably slow compared to the first cultivation phase. The second phase of the culture lasted an additional 70 to 80 hours. At this stage, human recombinant enterokinase accumulated significantly in the medium. The enzyme specific activity was in the range of 900 to 2500 U / ml culture medium filtrate. The volume of media obtainable in this way ranged from 850 to 1000 ml, which meant a total rhEK yield of 765 000 U to 2 500 000 U, which meant a total yield of the obtained enzyme of 700 000 U to 2 700 000 U rhEK calculated according to the commercial standard EKMax ™ (ThermoFisher Scientific).

Example 4:

Culturing P. pastoris to produce rhEKi. without an initial batch.

A culture of P. pastoris prepared according to Example 1 was replated to fresh YPD (1% yeast autolysate; 2% peptone; 2% glucose) agar plates and cultured at 29 ° C. Then, one isolated YPD colony was inoculated into 50 ml of inoculation medium (13.8 g / l (NH4) 2SO4, 46 g / l (NH4) H2PO4, 15.9 g / l KOH, 10 g / l glucose; 0.4 ml / l ( v / v) 1M MgSO4, 0.02 ml / l (v / v) 1M CaCl2, 4.6 ml / l PTM1, 10g / l yeast autolysate, 20 g / l peptone) in a 250 ml Erlenmayer flask and cultured for 24 hours at temperature 29 ° C. A solution of microelements and growth hormone PTM1: 0.5 g / l CoCl ₂ .6Ha, 65 g / l FeSO4.7H2O, 3 g / L MnSO4.5H ₂ O, 5 ml / l H2SO4 (95-98%), 0:08 g / L KI 6 g / l CuSO4.5H ₂ O, 20 g / l ZnCl _2, 0.02 g / l H3BO3, 0.2 g / l Na ₂ MoO4.2H ₂ O and 0.2 g of biotin.

The supplementary fermentation was carried out in a laboratory fermentor with a total volume of 2 liters at 29 ° C, using 750 ml of medium for cultivation: 26.7

-16ml / l H3PO4, 5.6ml / l H2SO4, 15.9g / l (w / v) KOH, 10g / l yeast autolysate and 20g / l peptone. After sterilization, the medium was supplemented with 4.6 ml / L PMT1. The feed medium was composed of 500 g / L glucose, 100 mM MgSO ₄ , 5 mM CaSO ₄ , and 12 mL / L PMT1. The culture conditions were controlled and maintained, pH 6.0 with 30% ammonium hydroxide, DOT to 60% saturation value by automatically increasing the stirrer speed, and manually adjusting the volume of aeration gases fed to the culture medium per unit time (vvm = volume of aeration per volume of medium per 1 minute), as well as manual feed medium dosing according to the specific biomass growth rate in the medium.

After reaching all the required parameters, a constant continuous feed rate of 0.1 ml / min was started from the beginning of the culture. and the fermentor was inoculated with 50 ml inoculum from a 24 hour culture of P. pastoris strain carrying on the chromosome the above construct with the gene encoding the human enterokinase light chain. At the beginning of the cultivation, the cells grew at the maximum specific growth rate and there was a partial accumulation of the carbon substrate in the medium, but the concentration at the start of the batch cultivation was about 20 g / L glucose. This procedure eliminated the inhibitory effect of too high a substrate concentration. After reaching a biomass concentration capable of consuming a pre-set feed rate, the feed medium dosage was set as in Example 2. This means that the feed rate was adjusted to the biomass growth rate. The dosing rate increased as long as the cells responded to the rate increase by increasing oxygen consumption. After reaching a feed rate at which the cells no longer responded, the feed rate was reduced and adjusted to a value at which the culture still reacted. Automatically with increasing demand for oxygen consumption by biomass, the mixing speed increased and the aeration manually increased. The aeration value increased at 500 rpm at 2 wm, at 1000 rpm at 3 wm and at 1500 rpm at 4 vvm. In this way, the specific growth rate was maintained above 0.05 h ¹ until 2000 rpm and 4 vvm aeration were achieved. Such a dosing method was maintained for 40 to 60 hours of culture.

After this phase of cultivation, the feed dosage was reduced so that the specific growth rate fell below 0.01 h * and the biomass growth in this phase was already considerably slow compared to the first cultivation phase. The second phase of the culture lasted 70-80 hours. During this phase, there was considerable accumulation of human recombinant enterokinase in the medium. The specific enzyme activity ranged from 1000 to 3000 U / ml culture medium filtrate. The volume of media obtainable in this manner ranged from 700 to 900 ml, representing a total yield of 700,000 U to 2,700,000 U rhEK, which is the activity expressed in international units, calculated according to the commercial standard EKMax ™ (ThermoFisher Scientific).

Human recombinant enterokinase (rhEK) was identified in the medium and isolated using affinity chromatography. The isolated enzyme was applied to SDS PAGE electrophoresis, where it was identified as a streak in the range of 70 to 130 kDa due to non-homogeneous glycosylation of enterokinase. After deglycosylation e.g. a strict band at the desired molecular weight (26.9 kDa) was identified by EndoHF. Further, the enzyme was tested for enzyme kinetics on a fluorogenic substrate containing the target amino acid sequence (GD4K-na), where the enzyme reached Km and kcat values comparable to those reported in recombinant human enterokinase production. In addition, the C-terminal sequence of 6 histidines was identified by Western blot. The identity of the human enterokinase light chain enzyme was confirmed by these methods

The cultivation method of the invention describes processes, methods and product yields allowing the preparation of commercial amounts of the human recombinant enterokinase enzyme.

Claims (9)

PATENT CLAIMS An expression DNA cassette carrying a gene encoding the human enterokinase light chain according to SEQ ID NO: 3. The expression DNA cassette according to claim 1, which carries a gene encoding the aMF secretion signal from Saccharomyces cerevisiae according to SEQ ID NO: 2, which is fused to the 5 'end of the gene encoding the human enterokinase light chain according to SEQ ID NO: 3. . An expression DNA cassette according to claim 2, which carries a promoter from the glyceraldehyde phosphate dehydrogenase gene according to SEQ ID NO: 1 operably upstream of the gene encoding the α-Mating Factor (aMF) secretion signal from Saccharomyces cerevisiae according to SEQ ID NO: 2, which is fused to the 5'-end of the gene encoding the human enterokinase light chain of SEQ ID NO: 3 in the same direction. The expression DNA cassette according to claim 3, which carries a promoter from the glyceraldehyde phosphate dehydrogenase gene according to SEQ ID NO: 1 operably upstream of the gene encoding the aMF secretion signal from Saccharomyces cerevisiae according to SEQ ID NO: 2 which is fused to 5'- the end of the gene encoding the human enterokinase light chain of SEQ ID NO: 3 in the same direction, wherein the sequences encoding the proteolytic cleavage sites for the Kex2 and Ste13 protease genes are located between the secretion signal coding sequence and the human enterokinase light chain gene of SEQ ID NO: 3 wherein the human enterokinase light chain, after proteolytic digestion at its N-terminus, contains only amino acid residues belonging to the human enterokinase light chain. A high-production strain Pichia pastoris carrying an expression cassette according to any one of claims 1 to 4 integrated in its chromosome. A method of cultivating a Pichia pastoris strain according to claim 5 for producing human enterokinase light chain, characterized in that the process is conducted in two phases, wherein in the first growth phase culture conditions are set to maintain a high specific biomass growth rate of the production strain which is at least 0.02 h ^-1 and in the second production phase, the culture conditions are set to maintain a low specific biomass growth rate of the production strain, which is at most 0.01 h -1. The cultivation method of claim 6, wherein the first growth phase of the cultivation is from 0 to 72 hours of cultivation and the conditions for the second production phase of cultivation are set within 24 to 36 hours after 72 hours of cultivation, wherein the second the phase lasts 60 to 80 hours. Cultivation method according to either of Claims 6 or 7, characterized in that in the first growth phase of the cultivation process control conditions are set in terms of aeration and agitation so as to achieve a high value of partial dissolved oxygen pressure (DOT) of 55 to 65 % and a controlled feeding process ensures a high specific biomass growth rate of the production strain, expressed as biomass dry weight (DCW) in the range of 150 to 200 g / L of culture medium, and significant accumulation occurs at a second specific production stage product of human recombinant enterokinase in the culture medium as a result of the high production rate of the culture of the production strain cultured under these conditions. The cultivation method according to claim 6, characterized in that in the first growth phase the cultivation conditions are set to maintain a high specific growth rate of the biomass of the production strain, which is at least 0.05 h -1.