阅读说明：本技术 一种重组胰蛋白酶的生产工艺 (A kind of production technology of recombinant trypsin ) 是由 乐峰松 于 2019-08-06 设计创作，主要内容包括：本发明提供了一种利用酵母重组表达可溶性重组胰蛋白酶的生产工艺,使胰蛋白酶能高表达于上清液中,并可利用离子交换层析方法从发酵液上清中纯化获得重组胰蛋白酶原,通过自身催化获得高活性重组胰蛋白酶,在成本、收率、活性和纯度等技术指标方面远远优于大肠杆菌表达工艺,因此极具工业化生产价值。(The present invention provides a kind of production technologies using yeast recombinant expression of soluble recombinant trypsin, trypsase height is set to be expressed in supernatant, and it is purified from fermented liquid supernatant using ion-exchange chromatography method and obtains recombinant trypsin original, high activity recombinant trypsin is obtained by autocatalysis, it is far superior to Bacillus coli expression technique, therefore great industrial production value in terms of the technical indicators such as cost, yield, activity and purity.)

1. a kind of production technology of recombinant trypsin, includes the following steps:

(1) after ultrafiltration or dilution being carried out containing the fermented liquid supernatant of recombinant trypsin original, then it is splined on ion-exchange chromatography Column；

(2) for ion exchange column first through Equilibration buffer wash, the equilibration buffer, which contains, can maintain the pH to be after loading The 5-100mmol/L buffer of 3.0-5.0,1-10mM CaCl₂；Ion gradient is carried out using elution buffer after rebalancing to wash De-, the elution buffer is that 0.1-2M sodium salt, collection of ions chromatographic elution target peak are added on the basis of equilibration buffer；

(3) elution target peak solution is adjusted to activate 10~30 hours under the conditions of pH7.0-11.0, keeps recombinant trypsin original living After turning to recombinant trypsin, then pH is adjusted to 2.5-5.0 and terminates activation；

(4) recombinant trypsin is precipitated by saltouing, be centrifuged, up to recombinant trypsin after freeze-drying.

2. production technology according to claim 1, it is characterised in that: the trypsase in the method is Recombinant Swine pancreas egg White enzyme, recombined bovine pancreas protease or recombination sheep trypsase.

3. production technology according to claim 1, it is characterised in that: the ion-exchange packing in the method step (1) For SP.

4. production technology according to claim 1, it is characterised in that: can maintain pH in the method step (2) is 3.0- 5.0 buffer can be optionally from disodium hydrogen phosphate-citrate buffer solution, phosphate buffer, Acetic acid-sodium acetate buffer or lemon Acid buffer.

5. production technology according to claim 4, it is characterised in that: in the method step (2), the equalizing and buffering Liquid contains 30-60mM NaAc-HAc, 2-4mM CaCl₂, pH 3.5-4.5.

6. production technology according to claim 1, it is characterised in that: in the method step (2), elution buffer be 0.1-2M sodium salt is added on the basis of equilibration buffer, the sodium salt is preferably sodium chloride, sodium sulphate and disodium hydrogen phosphate etc..

7. production technology according to claim 1, it is characterised in that: in the method step (3), prepared by step (2) Obtained ion chromatography target peak pH is adjusted to activate 15~20h under the conditions of 8.0-8.5, makes the activation of recombinant trypsin original for recombination After trypsase, then pH is adjusted to 2.5-3.5 and terminates activation.

8. production technology according to claim 1, it is characterised in that: in the method step (4), in the molten of step (3) Neutral salt is added in liquid precipitates trypsase, and the salt can be selected from ammonium sulfate, sodium sulphate, sodium chloride etc..

9. production technology according to claim 1, it is characterised in that: in the method step (4), terminate sample in activation Middle addition ammonium sulfate solids are to 0.7~1.5mol/L；After mixing evenly, it stands at room temperature, is centrifuged to obtain with tube centrifuge wet Solid；Wet solid is laid in pallet, obtains freeze-dried powder, as recombinant trypsin according to normal freeze-drying program.

10. production technology according to claim 1, it is characterised in that: the method further includes following steps:

(a), under the premise of keeping respective gene codon reading frame constant, will there is the fusion base shaped like A-B-C structure Because being connected to Yeast expression carrier, expression vector is converted and is imported in saccharomycete, obtains recombinant yeast through screening；Wherein, institute The nucleotide sequence that part A is encoding leader peptide is stated, B is the nucleotide sequence for encoding enterokinase enzyme site, and C is trypsase base Cause；

(b), high density fermentation recombinant yeast is 4.8-5.4 in growth phase control pH range, and induction period controls pH range For 3.2-3.6, fermented supernatant fluid is collected by centrifugation after expression.

Technical field

The present invention relates to field of biotechnology, and in particular to a kind of purifying production process of recombinant trypsin.

Background technique

The digestant that pancreatin system pharmacopoeia of each country records is the mixture of a variety of enzymes extracted from animal pancreas, mainly Ingredient is trypsase, pancreatic lipase and amylopsin.According to 2015 editions regulations of Chinese Pharmacopoeia, this strain is from pig, sheep or ox The mixture of a variety of enzymes extracted in pancreas, predominantly trypsase, amylopsin and pancreatic lipase.It is calculated by dry product, every lg In vigor containing trypsase must not be less than 600 units, amylopsin vigor must not be less than 7000 units, pancreatic lipase vigor must not Less than 4000 units.The production of pancreatin at present is mainly extracted by animal pancreas, is produced using existing pancreatin production technology, is produced Product pancreatin yield is lower, related process can refer to Chinese patent CN200910117583.5, CN201310124906.X and CN201310124907.4 etc., is hereby incorporated by reference.

In pancreatin component, trypsase is a kind of serine protease, the enzymatic in basic amino acids arginine and The hydrolysis cutting (Keil B., 1971) of peptide is carried out on the carboxylic group of lysine, there are many research shows that trypsase can be with It is separated from other higher vertebrates, such as ox, pig, sheep.The enzyme is as inactive precursor (trypsinogen) In It is synthesized in the pancreatic cell of vertebrate, and is then changed into active form and the cutting to propetide.First tryptose Plasminogen molecule is by the hydrolysising peptide key between (Asp4)-Lys- ↓-Ile in natural mode to cut off the erepsin intestines of propetide Kinases is come (Keil, 1971) that activates).Priming reaction can also autocatalytically carry out under physiological ph, this is because having one A lysine is located at the C- end side of the identification sequence of enterokinase, therefore Lys- ↓-Ile peptide bond hydrolysis can also pass through trypsase It carries out (Light et al., 1980).

Trypsase is mainly used for for polypeptide being cut into the segment for being used to be sequenced, is used to making attached cell from capped thin It is detached from born of the same parents' culture ware and for fused protein to be cut into target peptide and fusion ingredient, is used to activate propetide (such as pancreas egg White proenzyme is to trypsase) and the recombinant production (such as proinsulin to insulin, referring to WO99/10503) for peptide hormone etc.. Trypsase is since it is easy to obtain from a variety of mammals, high specific (is only carried out in lysine or the arginic end C- Cutting) and high specific activity (~150U/mg) and its good storage-stable, so had in biotechnology applications always The protease of value.

Since the use of the enzyme of animal origin has no longer been allowed in many cases (potential virus pollution etc.), because This in industrial application especially for needed in the production process of drug using provide the recombination pancreas egg from microbial hosts White enzyme.But since trypsase is the strong endopeptidase of activity, any lysine for being exposed to protein surface of hydrolyzable and The peptide bond that the c-terminus of arginine residues is formed, including its own, so general trypsase is unstable in pH3 or more, meeting Itself hydrolysis, and the presence of trace active trypsase can generate toxicity to expression host cell.A kind of solution is It is expressed in the form of trypsinogen, such as CN201610005823.2 is by going out bovine trypsinogen in expression in escherichia coli Or the inclusion body protein of bovine trypsinogen fusion protein, acidification is then carried out by inclusion body protein change, renaturation as folding Good bovine trypsinogen or bovine trypsinogen fusion protein.But since the technique is insoluble inclusion bodies, packet The renaturation yield for containing body is very low, and purifying process is relative complex, therefore the yield of the recombinant trypsin of its report is relatively low.

Therefore, new soluble recombinant trypsin production technology is developed, to obtain low cost, high yield and high enzyme activity Recombinant trypsin be still urgent technical problem to be solved in the field.

Summary of the invention

In view of the above-mentioned problems, the present invention provides a kind of productions using yeast recombinant expression of soluble recombinant trypsin Technique is enable the high expression of trypsase, is purified from fermented liquid supernatant using ion-exchange chromatography method and obtain recombination tryptose Proenzyme, and high activity recombinant trypsin is obtained by autocatalysis.

On the one hand, the invention discloses a kind of production technology of recombinant trypsin, include the following steps:

(1) after ultrafiltration or dilution being carried out containing the fermented liquid supernatant of recombinant trypsin original, then it is splined on ion exchange Chromatographic column；

(2) first through Equilibration buffer wash, the equilibration buffer contains to be maintained ion exchange column after loading PH is the 5-100mmol/L buffer of 3.0-5.0,1-10mM CaCl₂；Ion ladder is carried out using elution buffer after rebalancing Degree elution, the elution buffer are that 0.1-2M sodium salt, collection of ions chromatographic elution mesh are added on the basis of equilibration buffer Mark peak；

(3) elution target peak solution is adjusted to activate 10~30 hours under the conditions of pH7.0-11.0, makes recombinant trypsin Original activation be recombinant trypsin after, then by pH be adjusted to 2.5-5.0 terminate activation；

(4) recombinant trypsin is precipitated by saltouing, be centrifuged, up to recombinant trypsin after freeze-drying.

In another preferred embodiment of the present invention, the trypsase in the method is recombination Porcine trypsin, again Group bovine trypsin or recombination sheep trypsase etc..In another preferred embodiment of the present invention, the trypsase To recombinate Porcine trypsin.

In another preferred embodiment of the present invention, in the method step (1), fermented liquid supernatant first carry out ultrafiltration or Dilution pretreatment, preferably fermented liquid supernatant purify be diluted with water after loading again.

In another preferred embodiment of the present invention, the ion-exchange packing in the method step (1) be strong sun from Sub- filler.It is furthermore preferred that ion-exchange packing is SP.

In another preferred embodiment of the present invention, pH can be maintained in the equilibration buffer in the method step (2) It can be optionally from disodium hydrogen phosphate-citrate buffer solution, phosphate buffer, Acetic acid-sodium acetate buffer for the buffer of 3.0-5.0 Or citrate buffer solution etc..Preferred buffer concentration is 10-80mmol/L, pH 3.5-4.5.In reality specifically preferred according to the invention It applies in scheme, equilibration buffer contains 30-60mM NaAc-HAc, 2-4mM CaCl₂, pH 3.5-4.5.It is furthermore preferred that balance Buffer contains 40mM NaAc-HAc, 2mM CaCl₂,pH4.5.Ion exchange column in the method step (2) first passes through Equilibration buffer wash 1-3 column volume (CV) so that conductance walked with pH baseline it is flat；Chromatographic column is put down again with equilibration buffer after loading Weigh 1-3CV.

In another preferred embodiment of the present invention, in the method step (2), elution buffer is in equalizing and buffering 0.1-2M sodium salt is added on the basis of liquid, the sodium salt is preferably sodium chloride, sodium sulphate and disodium hydrogen phosphate etc..More preferably , the sodium salt is 0.4M sodium chloride.Preferred eluent is 40mM NaAc-HAc, 0.4M NaCl, 2mM CaCl2, pH4.5.Target peak is sampled into electrophoresis detection, purity can be 95% or more.

In another preferred embodiment of the present invention, after the completion of ion gradient elution, ion exchange column can be used first 0.5-2M NaOH rinses 1-2CV, then rinses 1-3CV with elution buffer, uses 1~2CV of Equilibration buffer wash, then with again Raw ion exchange column.

In another preferred embodiment of the present invention, in the method step (3), preferably step (2) is prepared Ion chromatography target peak pH be adjusted to activate 15~20h under the conditions of 8.0-8.5, make the activation of recombinant trypsin original for recombination pancreas egg After white enzyme, then pH is adjusted to 2.5-3.5 and terminates activation.It is furthermore preferred that the ion chromatography target peak that step (2) is prepared is used Tris buffer is adjusted to pH8.0 ± 0.2, after mixing evenly, activates 15~20h at room temperature；Sample HCl after activation Solution tune pH2.8 ± 0.2 is to terminate activation.

In another preferred embodiment of the present invention, in the method step (4), it is added in the solution of step (3) Neutral salt precipitates trypsase, and the salt can be selected from ammonium sulfate, sodium sulphate, sodium chloride etc..It is preferred that terminating sample in activation Ammonium sulfate solids are added in product to 0.7~1.5mol/L；After mixing evenly, it stands at room temperature, is centrifuged to obtain with tube centrifuge Wet solid.Wet solid is laid in pallet, obtains freeze-dried powder, as recombinant trypsin according to normal freeze-drying program. In another preferred embodiment, the enzyme activity of Porcine trypsin is prepared in measurement, as a result Porcine trypsin vigor >=2000- 5000USP/mg。

On the other hand, the present invention provides a kind of production works using yeast recombinant expression of soluble recombinant trypsin Skill is enable the high expression of trypsase, and is purified and recombinated from fermented liquid supernatant using ion-exchange chromatography method above-mentioned Trypsinogen, and high activity recombinant trypsin is obtained by autocatalysis.

It therefore, further comprise walking as follows on the other hand the invention discloses a kind of production technology of recombinant trypsin It is rapid:

(a), under the premise of keeping respective gene codon reading frame constant, will there is melting shaped like A-B-C structure It closes gene and is connected to Yeast expression carrier, expression vector is converted and is imported in saccharomycete, obtain recombinant yeast through screening；Its In, the part A is the nucleotide sequence of encoding leader peptide, and B is the nucleotide sequence for encoding enterokinase enzyme site, and C is pancreas egg White enzyme gene；

(b), high density fermentation recombinant yeast is 4.8-5.4 in growth phase control pH range, and induction period controls pH Range is 3.2-3.6, and fermented supernatant fluid is collected by centrifugation after expression.

In another preferred embodiment of the present invention, the above-mentioned fermented supernatant fluid being prepared can utilize ion above-mentioned The purifying of displacement chromatography method obtains recombinant trypsin original, is recombinant trypsin through autoactivation, purifying process parameter is such as It is preceding described.

Trypsase belongs to serine protease, carboxylic group of the enzymatic in basic amino acids arginine and lysine The upper hydrolysis cutting for carrying out peptide, there are ox, pig and sheep etc. in source.In a preferred embodiment of the present invention, the pancreas Protease is Porcine trypsin, and the Porcine trypsin original sequence of building is as shown in Seq ID No:1, the pig pancreas egg after autoactivation The amino acid sequence of white enzyme is as shown in Seq ID No:2.

In another preferred embodiment of the present invention, fusion (its sequence such as Seq ID of A-B-C structure is built Shown in No:3) after, it can be with method known in this field by the nucleic acid clone of the sequence containing encoding fusion protein to various expression vectors In.The molecular cloning process of standard used is shown in (J. Pehanorm Brooker etc., " Molecular Cloning:A Laboratory guide " such as J. Pehanorm Brooker The second edition, Science Press, 1995.) narration.Different fusions can express to obtain various fusion eggs in host respectively It is white.Many expression vectors host corresponding with its can be bought with subsidiary company, such as Yeast expression carrier pPICZ- α-A, pHIL-D2, PPIC9 and pHIL-S1 (Invitrogen Corp.San Diego.California.USA) etc., animal cell expression vectors PSVK3, pMSG (Amersham Pharmacia Biotech Inc.USA) etc..Preferred method will encode in the present invention For the nucleic acid clone of target enzyme to Yeast expression carrier pPICZ- α-A, which is yeast integrative plasmid, is grasped with alcohol oxidase Vertical son (AOX1) 5 ' sequence and 3 ' sequences, for facilitating encoding gene to be integrated into yeast chromosomal, and the table of control encoding gene It reaches.These plasmids and corresponding host strain etc. can be from Invitrogen Corp.San Diego.California.USA structures , preferred promoter is AOX1.

Carrier can be inverted or be transfected to prokaryotes or eucaryote host.Nucleic acid needed for converting is into host cell Usual way can be used, such as: electroporation prepares the spheroplast etc. of competence.Expression target protein host can be yeast, Mammalian cell, bacterium, animal, plant etc., preferably saccharomycete, as saccharomyces cerevisiae, Pichia pastoris, candida yeasts, Hansenula yeast, Crewe tie up sub- yeast, the female category yeast or fission yeast etc. of spore circle, more preferably Pichia pastoris.Target protein or enzyme It can reside in host cell, be also possible to be secreted from host, it is preferred that is secreted from host.Secretion Signal peptide used, preferably yeast MF alpha signal peptide.

The host that DNA construct of the present invention can be contained by cultivating, such as recombination yeast, recombinant mammalian cells, again Group bacterium etc., produces recombinant trypsin.In a preferred embodiment of the present invention, genetic engineering ferment building completed Female bacterium carries out Liquid Culture and fermentation, expresses target protein by methanol induction.The condition of fermentation culture stage controls are as follows: raw Long, induction period temperature control is at 20~30 DEG C；PH is controlled 4.0~5.0.In incubation, when dissolved oxygen persistently rises, need Add glycerine supplemented medium.After equal dissolved oxygens and pH rise, starts stream plus induction supplemented medium (glycerine+methanol) is opened Dynamic induction program, on the one hand adding glycerine can be improved cell density, on the other hand add methanol induction protein expression.In order to It allows saccharomycete to adapt to methanol, the feed rate of methanol can gradually be turned up stage by stage, induce the expression of target protein.Work as fermentation liquid Bacterium OD₆₀₀When value >=300, fermentation liquid is released, fermentation supernatant is collected by centrifugation.

Albumen can be isolated and purified with the method for various Protein Separations, such as saltout, precipitate, ultrafiltration, liquid chromatography technology And the combination of these technologies, wherein liquid chromatography can use the chromatographic techniques such as gel exclusion, affine, ion exchange, hydrophobic, reverse phase Or combinations thereof.It is preferred that obtaining recombinant trypsin original after purification through ion-exchange chromatography method, it is prepared through autoactivation Recombinant trypsin, purifying process parameter are as previously described.

Using recombinant trypsin purifying production process disclosed by the invention, purity >=95%, enzyme activity can be prepared The high-quality of >=2000-5000USP/mg recombinates Porcine trypsin.Method disclosed by the invention is recombinated using yeast secreted expression Trypsinogen avoids Bacillus coli expression from needing the complicated processes of renaturation, and technique is simpler, and in cost, yield, activity It is far superior to the prior art, therefore great industrial production value with technical indicators aspects such as purity.

Detailed description of the invention

Attached drawing 1: Yeast expression carrier constructs schematic diagram.

Attached drawing 2: (wave crest zigzag is UV280 absorption value to trypsinogen ion-exchange chromatography figure, ladder-like for solution electricity Lead value).

Attached drawing 3: trypsase electrophoretogram (12%SDS-PAGE), wherein band 1: molecular weight marker；Band 2:01 batches Trypsase；Band 3:02 batches of trypsase.

Specific embodiment