CN110511916B

CN110511916B - Production process of recombinant trypsin

Info

Publication number: CN110511916B
Application number: CN201910720623.9A
Authority: CN
Inventors: 乐峰松
Original assignee: Hangzhou Putai Biotechnology Co ltd
Current assignee: Hangzhou Putai Biotechnology Co ltd
Priority date: 2019-08-06
Filing date: 2019-08-06
Publication date: 2021-06-01
Anticipated expiration: 2039-08-06
Also published as: CN110511916A

Abstract

The invention provides a production process for recombinant expression of soluble recombinant trypsin by using yeast, which enables the trypsin to be highly expressed in supernatant, can purify and obtain recombinant trypsinogen from supernatant of fermentation liquor by using an ion exchange chromatography method, obtains the high-activity recombinant trypsin by autocatalysis, and is far superior to an escherichia coli expression process in the aspects of technical indexes such as cost, yield, activity, purity and the like, thereby having great industrial production value.

Description

Production process of recombinant trypsin

Technical Field

The invention relates to the technical field of biology, in particular to a purification production process of recombinant trypsin.

Background

The pancreatin is a digestant drug collected in pharmacopoeia of various countries, is a mixture of multiple enzymes extracted from animal pancreas, and mainly comprises trypsin, pancreatic lipase and pancreatic amylase. According to the regulation of Chinese pharmacopoeia 2015 edition, the product is a mixture of various enzymes extracted from pig, sheep or cattle pancreas, and mainly comprises trypsin, pancreatic amylase and pancreatic lipase. Calculated according to a dry product, each lg contains not less than 600 units of trypsin activity, not less than 7000 units of pancreatic amylase activity and not less than 4000 units of pancreatic lipase activity. At present, pancreatin is mainly produced by extracting animal pancreas and adopting the existing pancreatin production process, the yield of the pancreatin is lower, and related processes can refer to Chinese patents CN200910117583.5, CN201310124906.X, CN201310124907.4 and the like, which are all incorporated by reference.

Among the pancreatin components, trypsin is a serine protease that catalyzes the hydrolytic cleavage of peptides at the carboxyl groups of the basic amino acids arginine and lysine (Keil b., 1971), and various studies have shown that trypsin can be isolated from other higher vertebrates, such as cattle, pigs, sheep, etc. The enzyme is synthesized as an inactive precursor (trypsinogen) in vertebrate pancreatic cells and subsequently converted to the active form by cleavage of the propeptide. The first trypsinogen molecule is activated in a natural way by enteropeptidase enterokinase which cleaves the propeptide by hydrolysis of the peptide bond between (Asp4) -Lys- ↓ -Ile (Keil, 1971)). The activation reaction also proceeds autocatalytically at physiological pH, since there is a lysine located C-terminal to the recognition sequence of enterokinase, and thus Lys- ↓ -Ile peptide bond hydrolysis can also be performed by trypsin (Light et al, 1980).

Trypsin is mainly used for cleaving polypeptides into small fragments for sequencing, for detaching adherent cells from covered cell culture dishes and for cleaving fusion proteins into target peptides and fusion components, for activating pro-peptides (e.g. trypsinogen to trypsin) and for recombinant production of peptide hormones (e.g. pro-insulin to insulin, see WO99/10503), etc. Trypsin is always a valuable protease in biotechnological applications due to its ready availability from a variety of mammals, high specificity (cleavage only at the C-terminus of lysine or arginine) and high specific activity (-150U/mg) and its good storage stability.

Since the use of enzymes of animal origin is no longer permitted in many cases (potential viral contamination, etc.), the use of recombinant trypsin provided from a microbial host is required in industrial applications, in particular for the production of pharmaceuticals. However, since trypsin is a highly active endopeptidase that can hydrolyze any peptide bonds formed at the carboxyl ends of lysine and arginine residues exposed on the protein surface, including itself, typical trypsin is unstable above pH3, will itself hydrolyze, and the presence of trace amounts of active trypsin can be toxic to the expressing host cell. One solution is to express the protein in the form of trypsinogen, for example, CN201610005823.2 expresses the inclusion body protein of bovine trypsinogen or bovine trypsinogen fusion protein in Escherichia coli, and then the inclusion body protein is changed and restored into folded bovine trypsinogen or bovine trypsinogen fusion protein by acidification treatment. However, the process is in the form of insoluble inclusion bodies, so that the renaturation rate of the inclusion bodies is low, and the purification process is relatively complex, so that the reported yield of the recombinant trypsin is low.

Therefore, the development of a new production process of soluble recombinant trypsin to obtain recombinant trypsin with low cost, high yield and high enzyme activity is still an urgent technical problem to be solved in the field.

Disclosure of Invention

Aiming at the problems, the invention provides a production process for recombinant expression of soluble recombinant trypsin by using yeast, which enables the trypsin to be highly expressed, obtains recombinant trypsinogen by purifying supernatant fluid of fermentation liquor by using an ion exchange chromatography method, and obtains the high-activity recombinant trypsin by autocatalysis.

On one hand, the invention discloses a production process of recombinant trypsin, which comprises the following steps:

(1) ultrafiltering or diluting the fermentation broth supernatant containing the recombinant trypsinogen, and loading the fermentation broth supernatant onto an ion exchange chromatographic column;

(2) washing the ion exchange chromatography column with balance buffer solution containing 5-100mmol/L buffer solution capable of maintaining pH at 3.0-5.0 and 1-10mM CaCl₂(ii) a After rebalancing, carrying out ion gradient elution by using an elution buffer solution, wherein the elution buffer solution is prepared by adding 0.1-2M sodium salt on the basis of the balance buffer solution and collecting an ion chromatography elution target peak;

(3) adjusting the pH value of the elution target peak solution to 7.0-11.0, activating for 10-30 hours to activate the recombinant trypsin to recombinant trypsin, and adjusting the pH value to 2.5-5.0 to terminate activation;

(4) precipitating the recombinant trypsin by salting out, centrifuging, and freeze-drying to obtain the recombinant trypsin.

In another preferred embodiment of the present invention, the trypsin in the method is recombinant porcine trypsin, recombinant bovine trypsin or recombinant ovine trypsin, etc. In another preferred embodiment of the invention, the trypsin is recombinant porcine trypsin.

In another preferred embodiment of the present invention, in step (1), the fermentation broth supernatant is subjected to ultrafiltration or dilution pretreatment, and preferably, the fermentation broth supernatant is diluted with purified water and then loaded.

In another preferred embodiment of the present invention, the ion exchange filler in step (1) of the process is a strong cationic filler. More preferably, the ion exchange packing is SP.

In another advantage of the inventionIn selected embodiments, the buffer capable of maintaining a pH of 3.0 to 5.0 in the equilibration buffer in step (2) of the method may optionally be selected from disodium hydrogen phosphate-citric acid buffer, phosphate buffer, acetic acid-sodium acetate buffer, citric acid buffer, or the like. Preferably, the buffer concentration is 10-80mmol/L and the pH is 3.5-4.5. In a particularly preferred embodiment of the invention, the equilibration buffer contains 30-60mM NaAc-HAc,2-4mM CaCl₂The pH value is 3.5-4.5. More preferably, the equilibration buffer contains 40mM NaAc-HAc,2mM CaCl₂pH 4.5. Washing the ion exchange chromatography column in the step (2) of the method by 1-3 Column Volumes (CV) with an equilibrium buffer solution to level the conductance and the pH base line; after loading, the column is re-equilibrated with equilibration buffer for 1-3 CV.

In another preferred embodiment of the present invention, in step (2), the elution buffer is prepared by adding 0.1-2M sodium salt, preferably sodium chloride, sodium sulfate, disodium hydrogen phosphate, etc., to the equilibration buffer. More preferably, the sodium salt is 0.4M sodium chloride. A preferred eluent is 40mM NaAc-HAc,0.4M NaCl,2mM CaCl2, pH 4.5. The purity can be over 95 percent by sampling the target peak for electrophoretic detection.

In another preferred embodiment of the present invention, after the ion gradient elution is completed, the ion exchange chromatography column may be regenerated by washing the column with 0.5-2M NaOH for 1-2CV, then washing the column with an elution buffer for 1-3CV, and then washing the column with an equilibration buffer for 1-2 CV.

In another preferred embodiment of the present invention, in the step (3), the ion chromatography prepared in the step (2) is preferably activated for 15-20 h under the condition that the pH of the target peak is adjusted to 8.0-8.5, and after the recombinant trypsin is activated from the recombinant trypsin origin, the pH is adjusted to 2.5-3.5 to terminate the activation. More preferably, the ion chromatography target peak prepared in the step (2) is adjusted to pH8.0 +/-0.2 by using a Tris buffer, and after being uniformly stirred, the ion chromatography target peak is activated for 15-20 hours at room temperature; the activated sample was adjusted to pH 2.8. + -. 0.2 with HCl solution to stop the activation.

In another preferred embodiment of the present invention, in step (4) of the method, trypsin is precipitated by adding a neutral salt to the solution of step (3), said salt being selected from ammonium sulfate, sodium chloride, and the like. Preferably, ammonium sulfate solid is added into the activation termination sample to 0.7-1.5 mol/L; after being stirred uniformly, the mixture is stood at room temperature and centrifuged by a tubular centrifuge to obtain wet solid. And flatly paving the wet solid in a tray, and obtaining freeze-dried powder, namely the recombinant trypsin, according to a conventional freeze-drying procedure. In another preferred embodiment, the enzyme activity of the prepared porcine trypsin is determined, resulting in a porcine trypsin activity of 2000-5000 USP/mg.

On the other hand, the invention provides a production process for recombinant expression of soluble recombinant trypsin by using yeast, which enables the trypsin to be highly expressed, and the recombinant trypsinogen is obtained by purifying the supernatant of fermentation liquor by using the ion exchange chromatography method and the high-activity recombinant trypsin is obtained by autocatalysis.

Therefore, in another aspect, the present invention discloses a process for producing recombinant trypsin, further comprising the steps of:

(a) under the premise of keeping the codon reading frame of each gene unchanged, connecting the fusion gene with the structure like A-B-C to a yeast expression vector, transforming the expression vector into yeast, and screening to obtain recombinant yeast; wherein, the part A is a nucleotide sequence for coding a leader peptide, the part B is a nucleotide sequence for coding an enterokinase restriction site, and the part C is a trypsin gene;

(b) and high-density fermentation of recombinant saccharomycete, controlling pH in the growth stage to 4.8-5.4 and pH in the induction stage to 3.2-3.6, expressing and centrifuging to collect the fermented supernatant.

In another preferred embodiment of the present invention, the fermentation supernatant obtained by the above preparation can be purified by the aforementioned ion exchange chromatography method to obtain recombinant trypsinogen, which is self-activated into recombinant trypsin, and the purification process parameters are as described above.

Trypsin belongs to the group of serine proteases which catalyse the hydrolytic cleavage of peptides at the carboxyl groups of the basic amino acids arginine and lysine, and is derived from cattle, pigs and sheep, among others. In a preferred embodiment of the invention, the trypsin is porcine trypsin, and the porcine trypsinogen sequence is constructed as shown in Seq ID No: 1, the amino acid sequence of the porcine trypsin after self-activation is shown as Seq ID No: 2, respectively.

In another preferred embodiment of the present invention, after constructing the fusion gene of the A-B-C structure (the sequence of which is shown in Seq ID No: 3), the nucleic acid containing the sequence encoding the fusion protein can be cloned into various expression vectors by methods known in the art. Standard molecular cloning procedures used are described in J. SammBruk et al (J. SammBruk et al, second edition of the molecular cloning, A laboratory Manual, science publishers, 1995). Different fusion genes can be expressed in a host respectively to obtain various fusion proteins. Many expression vectors and their corresponding hosts are commercially available from companies such as yeast expression vectors pPICZ-alpha-A, pHIL-D2, pPIC9 and pHIL-S1(Invitrogen Corp. san Diego. California. USA), animal cell expression vectors pSVK3, pMSG (Amersham Inc Pharmacia Biotech. USA), and the like. A preferred method is to clone a nucleic acid encoding the enzyme of interest of the present invention into a yeast expression vector pPICZ-alpha-A, which is a yeast integration type plasmid having 5 'and 3' sequences of the alcohol oxidase operon (AOX1) for facilitating integration of the encoding gene into the yeast chromosome and controlling the expression of the encoding gene. These plasmids and the corresponding host bacteria can be constructed from Invitrogen Corp.san Diego.Calif. USA, and the preferred promoter is AOX 1.

The vector may be transformed or transfected into a prokaryotic or eukaryotic host. Transformation of the desired nucleic acid into a host cell can be carried out by conventional methods, such as: electroporation, preparation of competent spheroplasts, and the like. The host for expressing the target protein can be yeast, mammalian cell, bacteria, animal, plant, etc., preferably yeast, such as Saccharomyces cerevisiae, Pichia pastoris, Candida, Hansenula, Kluyveromyces, Torulopsis, or Schizosaccharomyces, etc., more preferably Pichia pastoris. The protein or enzyme of interest may be present in the host cell or secreted from the host, preferably secreted from the host. The signal peptide used for secretion is preferably the yeast MF α signal peptide.

Can be obtained by culturing a host containing the DNA construct of the invention, e.g.Recombinant yeast, recombinant mammalian cells, recombinant bacteria, and the like, to produce recombinant trypsin. In a preferred embodiment of the present invention, the constructed genetically engineered yeast is subjected to liquid culture and fermentation, and the target protein is expressed by methanol induction. The conditions of the fermentation culture stage are controlled as follows: controlling the temperature at 20-30 ℃ in the growth and induction stages; the pH is controlled to be 4.0-5.0. In the culture process, when the dissolved oxygen continuously rises, the glycerol feed culture medium needs to be supplemented. After dissolved oxygen and pH rise, starting a fed-batch induction supplemented medium (glycerol and methanol) to start an induction program, on one hand, supplementing glycerol can improve the thallus density, and on the other hand, supplementing methanol can induce protein expression. In order to adapt the yeast to methanol, the feeding speed of the methanol can be gradually increased in stages to induce the expression of the target protein. When fermentation broth bacteria OD₆₀₀When the value is more than or equal to 300, discharging the fermentation liquor, and centrifugally collecting fermentation supernatant.

The protein can be separated and purified by various methods of protein separation, such as salting out, precipitation, ultrafiltration, liquid chromatography, and combinations thereof, wherein the liquid chromatography can be performed by gel exclusion, affinity, ion exchange, hydrophobic, reverse phase, and other chromatographic techniques, or combinations thereof. Preferably, the recombinant trypsinogen is obtained after purification by an ion exchange chromatography method, and the recombinant trypsin is prepared through self-activation, wherein the purification process parameters are as described above.

The purification production process of the recombinant trypsin can be used for preparing the high-quality recombinant porcine trypsin with the purity of more than or equal to 95 percent and the enzyme activity of more than or equal to 2000-5000 USP/mg. The method disclosed by the invention utilizes yeast to secrete and express the recombinant trypsinogen, avoids the complicated process that escherichia coli expression needs renaturation, has simpler process, and is far superior to the prior art in the aspects of technical indexes such as cost, yield, activity, purity and the like, so that the method has great industrial production value.

Drawings

FIG. 1: schematic diagram of yeast expression vector construction.

FIG. 2 is a drawing: trypsinogen ion exchange chromatography (peak-saw tooth shape is UV280 absorbance, step-like is solution conductivity).

FIG. 3: trypsin electrophorogram (12% SDS-PAGE), wherein band 1: molecular weight marker; lane 2:01 batch trypsin; band 3:02 batch trypsin.

Detailed Description

Example 1

First, a gene sequence (shown as Seq ID No: 3) is translated according to a porcine trypsinogen sequence (shown as Seq ID No: 1), the 5 'end of the gene sequence contains a nucleic acid sequence corresponding to an EK enzyme cutting site and a 5' Xho I (ctcgag) enzyme cutting site, and the 3 'end of the gene sequence is added with a terminator and a 3' Not I (gcggccgc) enzyme cutting site. The above sequences were synthesized by the gene synthesis service company, cloned into pGEM-T (Promega corporation) vector, and verified by sequencing to be complete and correct. Then respectively extracting plasmids, and carrying out double enzyme digestion on PGEMT by Xho I/Not I to recover the gene of the porcine trypsin; the method comprises the steps of recovering a large fragment by using Xho I/Not I double enzyme digestion pPICZ alpha A yeast expression gene, then connecting the two fragments to form a recombinant yeast expression plasmid pPICZ alpha A containing a coding yeast alpha-factor leader peptide-enterokinase site-porcine trypsin gene form, verifying a reading frame by sequencing, and constructing a plasmid as shown in figure 1. The recombinant yeast expression vector is subjected to in vitro enzyme digestion linearization, and a Pichia pastoris acceptor strain GS115 is transformed by a protoplast transformation method. And (3) coating the transformant on an MD culture medium, culturing for 48h at 30 ℃, and selecting the transformant growing on the MD culture medium as a porcine trypsin engineering expression strain.

Example 2 expression fermentation of porcine pancreatin

The porcine trypsin engineering expression strain is picked by an inoculating loop and put into YPG medium (6 g of tryptone, 3 g of yeast powder and 6 g of glycerol are put in a 5L glass beaker, the volume is kept to 300 mL by purified water and the glass beaker is filled in 1 1000mL triangular flask). The inoculated YPG was shake-flask placed in a shaking table and cultured at 220rpm for about 18-24 hours so that OD 600. gtoreq.5 was used as seed liquid.

Pouring the seed liquid into a fermentation tank, supplementing yeast culture medium, and regulating aeration flow to be not less than 10L/min. The conditions of the fermentation culture stage are controlled as follows: controlling the temperature to be 20-30 ℃; the pH is controlled to be 4.0-5.0. Checking and recording for 1 time every 0.5-2 hours, wherein the recorded contents comprise: temperature, pH, dissolved oxygen PO2, stirring Stir, aeration Air, base content, aeration content, and supplement content. When the culture is carried out until the stirring speed is opened to the maximum, the oxygen valve is opened.

After about 24 hours of the culture, when the dissolved oxygen continued to rise, glycerol feed medium (glycerol 50% v/v, purified water 50% v/v) was started at a rate of 20 to 25 ml/L/h. After the glycerol feeding is finished, after dissolved oxygen and pH rise, the temperature is reduced to 25 ℃, the pH is set to 3.2, and an induction program is started. And starting to feed an induction supplemented medium (50% of glycerol and 50% of methanol), wherein on one hand, the glycerol is supplemented to improve the thallus density, and on the other hand, the methanol is supplemented to induce protein expression. To acclimate yeast to methanol, the feed rate of methanol was gradually increased in stages: 2mL/L/h for 0-2 hours, 4.0mL/L/h for 2-4 hours, and then 6.0 mL/L/h. The total induction is about 118-125 hours, and the fermentation liquor has the bacterial concentration OD₆₀₀Discharging the fermentation liquor when the fermentation liquor is more than or equal to 300 ℃, and centrifugally collecting fermentation supernatant.

Example 3 purification of porcine pancreatin

1) Sample pretreatment: adding 1 time of purified water into the fermentation supernatant, and finally, the conductivity of the sample liquid is about 7.0 ms/cm; the pH was controlled at about 4.5.

2) And (3) chromatographic column balancing: a chromatography column with a diameter of 25cm and an ion exchange column SP Bestarose FF (Bogelong Biotechnology Co., Ltd.). 3CV was equilibrated with equilibration buffer (50mM NaAc-HAc,2mM CaCl2, pH 4.5; conductivity leveled off with the pH baseline.

3) Loading: and (4) sampling the pretreated fermentation liquor.

4) Re-equilibration of the chromatographic column: equilibrate 2CV with equilibration buffer.

5) Sample elution: the target peak was eluted with an eluent (50mM NaAc-HAc,0.4M NaCl,2mM CaCl2, pH 4.5).

6) And (3) regenerating a chromatographic column: washing 1CV with 1mol/L NaOH, then washing 1CV with eluent, and then washing 1-2CV with balancing solution.

7) And (3) detection: sampling and detecting by electrophoresis, wherein the purity is over 97 percent.

8) And (3) sample preservation: and storing at room temperature.

The trypsinogen ion exchange chromatography is shown in FIG. 2.

Example 4 activation of porcine pancreatin

And (3) adjusting the pH value of the target peak of the ion chromatography prepared in the example 3 to 8.0 +/-0.2 by using 1mol/L Tris, uniformly stirring, and activating at room temperature for 15-20 h. The activated sample was adjusted to pH 4.0. + -. 0.2 with 50% HAc and then adjusted to pH 2.8. + -. 0.2 with 1mol/L HCl to stop the activation.

Adding 3.3M ammonium sulfate solution into the activated sample according to the volume ratio of 1: 2.8; after being stirred evenly, the mixture is stood for 3 hours at room temperature, and then centrifuged by a tubular centrifuge at 14000rpm and 4 ℃ at the injection speed of 60L/h to obtain wet solid. Spreading the wet solid in a tray, pre-freezing at-40 deg.C for 2 hr, heating to-4 deg.C, maintaining for 10 hr, and finally heating to 25 deg.C, maintaining for 8 hr to obtain lyophilized powder, i.e. porcine trypsin (sequence shown in Seq ID No: 2).

Example 5 analysis of purity of porcine pancreatin

The porcine trypsin prepared in example 2-4 was subjected to SDS polyacrylamide gel electrophoresis to check the purity. Mixing the sample to be detected with 5 times of reduction sample-adding buffer solution according to the ratio of 4:1, uniformly mixing, heating on a metal constant-temperature heater at 99.9 ℃, centrifuging at 10000rpm, and loading each sample by 5-10 mu l after centrifuging. Wherein each lane sample is strip 1: molecular weight marker; strip 2: batch 01 Trypsin; the strip 3: batch 02 trypsin.

After sample application, the power supply is switched on, electrophoresis is carried out for 30 minutes under the condition of 80V voltage, then the voltage is adjusted to 150V voltage for continuous electrophoresis, and the electrophoresis is stopped when the dye front reaches a position 0.5cm away from the bottom of the separation gel. And (3) adding an appropriate amount of fixing solution into the unloaded gel, fixing for 10min, adding an appropriate amount of staining solution, and gently shaking and staining for 1 hour. And (3) washing residual dyeing liquid by using tap water, adding a proper amount of decoloring liquid, shaking for 1-2 hours on a decoloring shaking table, replacing the decoloring liquid for several times until a blank background of the gel is basically colorless, and taking a picture of the gel, wherein the picture is shown in attached figure 3. Lanes and bands were analyzed with ImageLab software and molecular weights were calculated from standards. Wherein the porcine trypsin in

lanes

2 and 3 has a molecular weight of 20-30kDa and a purity of more than 97%.

Example 6 Activity analysis of porcine pancreatin

Trypsin can hydrolyze N-benzoyl-L-arginine ethyl ester (BAEE) as substrate to generate N-benzoyl-L arginine (BA) with higher absorption value at ultraviolet A253 nm. Under certain conditions, as the catalytic reaction progresses, the BAEE is gradually reduced, the BA products are gradually increased, the ultraviolet absorption value of the reaction system is gradually increased, and finally the delta A253nm is increased by 0.001 enzyme amount of 1 BAEE unit per minute (1USP/mg equals to 3 BAEE/mg).

A1 cm path length covered quartz cuvette was taken and 6.0ml of 0.25mM BAEE substrate working solution preheated to 25 ℃ was added and immediately zeroed at A253 nm. 5ul of 1mg/ml enzyme solution to be tested was added, mixed immediately and read. The reading was performed every 30s for a total of 6 min. The measured light absorption value delta 253nm/min is preferably kept between 0.02 and 0.03, and the added enzyme amount is to keep the delta 253nm/min consistent within 3 to 4 min. And (3) by taking time (t) as an abscissa and the light absorption value (delta 253nm) as an ordinate, constructing a graph, and optionally selecting a time interval (with a stable value of at least 3-4 minutes) and a corresponding light absorption value change (delta 253nm) in a straight line part to calculate a slope, namely obtaining delta 253 nm/min. The total number of the measurement is 3, and the average value of the 3 times is taken to calculate the activity of the pancreatin to be measured.

Trypsin activity unit (USP/mg pro) ═ Δ 253nm/min/(0.003 ═ enzyme solution addition volume)/protein content

According to the steps, the porcine trypsin prepared in the embodiment 2-4 is used for measuring the enzyme activity, and the result shows that the activity of the porcine trypsin is more than or equal to 5000USP/mg pro.

Sequence listing

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Claims

1. A production process of recombinant trypsin comprises the following steps:

(1) under the premise of keeping the codon reading frame of each gene unchanged, connecting the fusion gene with the structure like A-B-C to a yeast expression vector, transforming the expression vector into yeast, and screening to obtain recombinant yeast; wherein, the part A is a nucleotide sequence for coding a leader peptide, the part B is a nucleotide sequence for coding an enterokinase restriction site, and the part C is a trypsin gene; wherein the trypsin is porcine trypsin, and the fusion gene sequence of the A-B-C structure is shown as Seq ID No: 3 is shown in the specification;

(2) high-density fermentation of recombinant yeast, controlling pH range at 4.8-5.4 in growth stage and pH range at 3.2-3.6 in induction stage, expressing, centrifuging and collecting fermentation supernatant;

(3) ultrafiltering or diluting the fermentation broth supernatant containing the recombinant trypsinogen, and loading the fermentation broth supernatant onto an ion exchange chromatographic column;

(4) washing the loaded ion exchange chromatography column with an equilibration buffer solution containing 30-60mM NaAc-HAc and 2-4mM CaCl₂The pH value is 3.5-4.5; after rebalancing, carrying out ion gradient elution by using an elution buffer solution, wherein the elution buffer solution is prepared by adding 0.1-2M sodium salt on the basis of the balance buffer solution and collecting an ion chromatography elution target peak;

(5) adjusting the pH value of the elution target peak solution to 8.0-8.5, activating for 15-20 hours to activate the recombinant trypsin to recombinant trypsin, and adjusting the pH value to 2.5-3.5 to terminate activation;

(6) precipitating the recombinant trypsin by salting out, centrifuging, and freeze-drying to obtain the recombinant trypsin with the purity of more than 97 percent and the trypsin activity of more than or equal to 5000 USP/mg.

2. The production process according to claim 1, characterized in that: in the step (3), the supernatant of the fermentation liquor is subjected to ultrafiltration or dilution pretreatment.

3. The production process according to claim 2, characterized in that: in the step (3), the supernatant of the fermentation liquid is diluted with purified water and then loaded.

4. The production process according to claim 1, characterized in that: the ion exchange packing in step (3) of the method is a strong cation packing.

5. The production process according to claim 4, characterized in that: the ion exchange packing in step (3) of the process is SP.

6. The production process according to claim 1, characterized in that: in step (4) of the method, the equilibration buffer contains 40mM NaAc-HAc,2mM CaCl₂,pH4.5。

7. The production process according to any one of claims 1 to 6, characterized in that: washing the ion exchange chromatography column in the step (4) of the method by 1-3 Column Volumes (CV) with an equilibrium buffer solution to level the conductance and the pH base line; after loading, the column is re-equilibrated with equilibration buffer for 1-3 CV.

8. The production process according to claim 1, characterized in that: in the step (4), the elution buffer solution is prepared by adding 0.1-2M sodium salt on the basis of the equilibrium buffer solution, wherein the sodium salt is sodium chloride, sodium sulfate or disodium hydrogen phosphate.

9. The production process according to claim 8, characterized in that: in step (4) of the process, the sodium salt is 0.4M sodium chloride.

10. The production process according to claim 8, characterized in that: in step (4), the elution buffer is 40mM NaAc-HAc,0.4M NaCl,2mM CaCl2, pH 4.5.

11. The production process according to any one of claims 8 to 10, characterized in that: after ion gradient elution in the step (4) of the method is finished, the ion exchange chromatographic column is washed by 0.5-2M NaOH for 1-2CV, then washed by elution buffer solution for 1-3CV, and then washed by balance buffer solution for 1-2CV, so that the ion exchange chromatographic column is regenerated.

12. The production process according to claim 1, characterized in that: in the step (5), the ion chromatography target peak prepared in the step (4) is adjusted to pH8.0 +/-0.2 by using a Tris buffer solution, and after being uniformly stirred, the ion chromatography target peak is activated for 15-20 hours at room temperature; the activated sample was adjusted to pH 2.8. + -. 0.2 with HCl solution to stop the activation.

13. The production process according to claim 1, characterized in that: in step (6), adding a neutral salt selected from ammonium sulfate, sodium sulfate or sodium chloride to the solution of step (5) to precipitate trypsin.

14. The production process according to claim 13, characterized in that: in the step (6), ammonium sulfate solid is added into the activation termination sample to 0.7-1.5 mol/L; after being stirred uniformly, the mixture is stood at room temperature and centrifuged by a tubular centrifuge to obtain wet solid; and flatly paving the wet solid in a tray, and obtaining freeze-dried powder, namely the recombinant trypsin, according to a conventional freeze-drying procedure.

15. The production process according to claim 1, characterized in that: the expression vector in step (1) of the method is selected from the group consisting of yeast expression vectors pPICZ-alpha-A, pHIL-D2, pPIC9 and pHIL-S1.

16. The production process according to claim 1, characterized in that: in the step (1), the host for expressing the target protein is yeast selected from Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pichia pastoris (Pichia pastoris), Candida (Candida), Hansenula (Hansenula), Kluyveromyces (Kluyveromyces), Torulaspora (Tourlaspora), and Schizosaccharomyces (Schizosaccharomyces).

17. The production process according to claim 16, characterized in that: the yeast in the step (1) of the method is Pichia pastoris.

18. The production process according to claim 1, characterized in that: in the step (2), the constructed genetic engineering yeast is subjected to liquid culture and fermentation, target protein is expressed by methanol induction, and the conditions in the fermentation culture stage are controlled as follows: controlling the temperature at 20-30 ℃ in the growth and induction stages; controlling the pH value to be 4.0-5.0; in the culture process, when the dissolved oxygen continuously rises, glycerol is required to be supplemented to supplement the culture medium; after the dissolved oxygen and pH rise, the fed-batch induction medium glycerol + methanol is started to start the induction program.

19. The production process according to claim 1, characterized in that: in the step (2), in order to make the yeast adapt to the methanol, the feed supplement speed of the methanol is gradually increased in stages, and the expression of the target protein is induced.