WO2018021979A2 - Cassette d'expression d'adn portant un gène codant une chaîne légère d'entérokinase humaine, souche pichia pastoris comprenant ladite cassette d'adn, et son procédé de culture - Google Patents

Cassette d'expression d'adn portant un gène codant une chaîne légère d'entérokinase humaine, souche pichia pastoris comprenant ladite cassette d'adn, et son procédé de culture Download PDF

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WO2018021979A2
WO2018021979A2 PCT/SK2017/050005 SK2017050005W WO2018021979A2 WO 2018021979 A2 WO2018021979 A2 WO 2018021979A2 SK 2017050005 W SK2017050005 W SK 2017050005W WO 2018021979 A2 WO2018021979 A2 WO 2018021979A2
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cultivation
enterokinase
production
phase
light chain
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WO2018021979A3 (fr
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Ján KRAHULEC
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Univerzita Komenského v Bratislave
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21009—Enteropeptidase (3.4.21.9), i.e. enterokinase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • DNA Expression Cassette Carrying Gene Encoding Human Enterokinase Light Chain, Pichia pastoris Strain Comprising said DNA Cassette, and its Cultivation Method
  • 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.
  • 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).
  • enterokinases 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).
  • 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.
  • 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).
  • 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.
  • strain MutS 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the subject matter of this invention is formed also by the high-production Pichia
  • 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.
  • 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.
  • 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.
  • the conditions for the process control 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.
  • DOT partial dissolved oxygen tension
  • DCW dry biomass weight
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Example 1 Preparation of the production strain for rhEKi.
  • GAP glyceraldehyde dehydrogenase
  • 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.
  • the plasmid was linearized by restriction endonuclease ⁇ and purified by a DNA fragments purification kit.
  • the aliquot of 50 ⁇ electrocompetent cells was transformed by the linearized plasmid in total amount of 5 ⁇ g and the transformed cells were selected on the solid YPD (1 % yeast autolysate; 2% peptone; 2% glucose) medium supplemented with zeocin (100 ⁇ g/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.
  • 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 test for human enterokinase activity was performed by the application of the medium from the tested clones to 20 ⁇ 9 of the protein substrate carrying the enterokinase cleavage site in 5 ⁇ 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 inoculation medium (13.8 g/l (NH 4 ) 2
  • 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.
  • 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.
  • vvm volume of aeration per volume of medium
  • the feeding rate was reduced to the last value, at which the culture still reacted by the increased consumption of dissolved oxygen.
  • 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.
  • 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.
  • 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 EKMaxTM (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.
  • 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 inoculation medium (13.8 g/l (NH 4 ) 2 S0
  • 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.
  • 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 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 EKMaxTM (ThermoFisher Scientific).
  • 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.
  • the inoculation medium (13.8 g/l (NH 4 ) 2 S0 4
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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 EKMaxTM (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.
  • the strict band was identified at the level of the desired molecular weight (26.9 kDa).
  • 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.

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Abstract

L'invention concerne une cassette d'expression d'ADN constituée d'au moins la région promotrice d'une levure, après laquelle la séquence nucléotidique codant le signal de sécrétion est liée de manière fonctionnelle, la séquence nucléotidique codant un site approprié pour le traitement protéolytique de coupure du signal de sécrétion. En outre, la cassette d'expression d'ADN porte, après le signal de sécrétion, une séquence nucléotidique codant la chaîne légère de l'entérokinase humaine liée de manière fonctionnelle après le signal de sécrétion, comportant 6 histidines situées après l'extrémité C-terminale du gène de l'entérokinase. Ladite cassette d'expression d'ADN est intégrée dans le génome de la levure Pichia pastoris. L'invention concerne également le procédé de culture de ladite levure Pichia pastoris produisant l'entérokinase humaine recombinée. Le procédé de culture est basé sur la culture en deux phases dans le fermenteur : lors de la première phase, les cellules sont cultivées à un taux spécifique élevé de croissance de biomasse, lorsque la biomasse augmente de manière significative, et l'entérokinase ne s'accumule pas dans le milieu de culture. Cette première phase est immédiatement suivie par la seconde phase du procédé de culture : celle-ci est effectuée à un taux spécifique de croissance de biomasse plus faible, qui est représenté par une croissance minimale de la biomasse, mais en revanche, l'entérokinase est produite dans le milieu de culture et s'y accumule.
PCT/SK2017/050005 2016-07-29 2017-07-27 Cassette d'expression d'adn portant un gène codant une chaîne légère d'entérokinase humaine, souche pichia pastoris comprenant ladite cassette d'adn, et son procédé de culture WO2018021979A2 (fr)

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SK50048-2016A SK500482016A3 (sk) 2016-07-29 2016-07-29 Expresná DNA kazeta nesúca gén kódujúci ľahký reťazec ľudskej enterokinázy, kmeň Pichia pastoris nesúci uvedenú expresnú DNA kazetu a spôsob jeho kultivácie
SKPP50048-2016 2016-07-29

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012071257A1 (fr) 2010-11-23 2012-05-31 Allergan, Inc. Compositions et procédés de production d'entérokinase dans des levures
US20150020238A1 (en) 2012-04-06 2015-01-15 Nbm Co., Ltd. Plant producing human enterokinase light chain protein and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2221358A1 (fr) * 2009-02-24 2010-08-25 Universität für Bodenkultur Wien Levures prototrophiques de biotine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012071257A1 (fr) 2010-11-23 2012-05-31 Allergan, Inc. Compositions et procédés de production d'entérokinase dans des levures
US8557558B2 (en) 2010-11-23 2013-10-15 Allergan, Inc. Compositions and methods of producing enterokinase in yeast
US20150020238A1 (en) 2012-04-06 2015-01-15 Nbm Co., Ltd. Plant producing human enterokinase light chain protein and uses thereof

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