CN104312933A - Method for optimizing signal peptide and improving exocytosis expression of trypsin - Google Patents
Method for optimizing signal peptide and improving exocytosis expression of trypsin Download PDFInfo
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- CN104312933A CN104312933A CN201410555749.2A CN201410555749A CN104312933A CN 104312933 A CN104312933 A CN 104312933A CN 201410555749 A CN201410555749 A CN 201410555749A CN 104312933 A CN104312933 A CN 104312933A
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- 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)
- C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
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- 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
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21004—Trypsin (3.4.21.4)
Abstract
The invention discloses a method for optimizing a signal peptide and improving exocytosis expression of trypsin, belonging to the technical field of genetic engineering. The signal peptide is fused with trypsin to express 8 types of pichia pastoris exocytosis signal peptides, and a starter (pAOX) is induced by methanol to express regrouped trypsin on pichia pastoris GS115 chromosome. Compared with the original alpha-factor signal peptide, the enzyme activity of the trypsin of the alpha mf signal peptide is improved by 2.75 times, and the problem that the exocytosis amount of the trypsin is low can be solved. By utilizing the method to produce the trypsin, the yield is high, the process is simplified, and the industrial application is convenient to realize.
Description
Technical field
The present invention relates to a kind of method optimized signal peptide raising trypsinase exocytosis and express, belong to gene engineering technology field.
Background technology
Trypsinase, as a kind of important kind being comparatively early found to apply in serine protease, is found in mammiferous enteron aisle Digestive system the earliest.Commercial trypsinase is many to be extracted from mammalian pancreatic, owing to containing abundant proteolytic enzyme in pancreas, brings difficulty to trypsinase separating-purifying.In addition, the trypsinase in Mammals source mostly is trypsin mixture, medical applications exists and endangers the immunogenicity of human body.
In recent years, the trypsinase of animal-origin is expressed in intestinal bacteria, pichia spp by genetic engineering means, but escherichia coli expression often occurs inclusion body, pichia spp zymogen expression pig, bovine trypsin need enteropeptidase, the trypsinase Activation In Vitro of external source.SGT research, Koo-Bon-Joon etc. utilize streptomycete to commonly use the conventional pWHM3 plasmid (Ermp of host Streptomyces lividans1326 and streptomycete, erythromycin promotor) obtain in the recombinant trypsin belonging to secreting, expressing in bacterium together, this plasmid is transferred to can produce in tryptic streptomyces griseus Streptomyces griseus IFO13350 and expresses by Chi-Won-Jae etc., and streptomycete trypsin is expressed in E.coli system with inclusion bodies by the people (1998) such as Daisuke Nohara.Existing research have expressed the trypsinase in streptomyces griseus source in pichia spp with trypsinase form.
Heterogenous expression trypsinase distinct issues are, expressing quantity is low, trypsinase enzyme is lived low.Therefore, building and be used for pichia spp exocytosis signal peptide, improve trypsinase enzymatic activities, changing into product demand and reducing production cost significant for meeting trypsinase industry.
Summary of the invention
The invention provides the tryptic recombination microzyme of a kind of product, be nucleotide sequence be before the trypsase gene of sequence shown in SEQ ID NO.9 fusion nucleus nucleotide sequence be sequence shown in SEQ ID NO.7 signal peptide formed recombination, recombination is carried out in Host Strains express the recombination microzyme obtained.
Described signal peptide, its nucleotides sequence is classified as any one of sequence shown below: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.
Described signal peptide, in one embodiment of the invention, its nucleotide sequence is the sequence shown in SEQIDNO.7.
The Host Strains that sets out of described recombination microzyme be following any one: pichia pastoris GS115, pichia pastoris KM71, pichia pastorisX-33, pichia pastoris SMD1168.
Described recombination microzyme, in one embodiment of the invention, its Host Strains that sets out is pichia pastoris GS115.
The present invention also provides a kind of construction process of described recombination microzyme, before nucleotide sequence is the trypsase gene of the sequence shown in SEQ ID NO.9, add the signal peptide formation recombination that nucleotide sequence is the arbitrary shown sequence of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8, this recombination is connected in expression plasmid and forms recombinant plasmid, then be transformed in yeast and obtain recombinant bacterium.
The construction process of described recombinant bacterium is specific as follows: according to the trypsase gene (mt) that derive from streptomyces griseus (Streptomyces griseus) of nucleotide sequence as shown in SEQ ID NO.9 of synthesis, the 8 kind signal peptides of nucleotide sequence as shown in SEQ ID NO.1 to SEQ ID NO.8 (called after α as respectively, Sas, Ipr, Iss, Lss, nsB, α mf, Kps) primer is designed, recombination (α as-mt is obtained before adopting two-step pcr method to add signal peptide to trypsase gene respectively, Sas-mt, Ipr-mt, Iss-mt, Lss-mt, nsB-mt, α mf-mt, Kps-mt), described recombination is connected to yeast expression vector, obtains recombinant plasmid, described recombinant plasmid electricity is transformed pichia spp Pichia pastoris GS115, obtain recombination yeast engineering strain.
Described recombination be adopt two-step pcr method to obtain containing signal peptide sequence and trypsase gene sequence recombination.
Described expression plasmid be following any one: pGAPZA, pAO815, pGAP α A, pPIC9K, pPICZB.
Described expression vector is pPIC9K plasmid in one embodiment of the invention.
Described expression vector and Host Strains are purchased from Invitrogen company.
The present invention also provides a kind of method improving trypsinase exocytosis and express, and is use the recombination microzyme of above-mentioned structure to ferment, expresses trypsinase.
The method that described raising trypsinase exocytosis is expressed, in one embodiment of the invention, recombination amf-mt is obtained before adding the signal peptide of nucleotide sequence as shown in SEQ ID NO.7 the trypsase gene of nucleotide sequence as shown in SEQ ID NO.9 to, recombination obtains recombinant plasmid after transforming yeast expression vector, recombinant plasmid transformed is in pichia spp, namely obtain recombinant bacterium, express.
The method that described raising trypsinase exocytosis is expressed, in one embodiment of the invention, specifically: (1) composition sequence is the gene fragment of sequence shown in SEQ ID NO.9; (2) with the gene fragment of step 1 for template, use sequence is SEQ ID NO.11, the primer shown in SEQ ID NO.12 carries out PCR, as template after PCR primer reclaims, with sequence be SEQ ID NO.10, the primer shown in SEQ ID NO.12 carries out PCR and obtains recombination; (3) recombination obtained by NotI, BamH I double digestion step 2 and pPIC9K plasmid, connect with ligase enzyme and obtain recombinant plasmid; (4) recombinant plasmid transformed step 3 obtained, in pichia pastoris GS115, namely obtains recombinant yeast pichia pastoris bacterium, expresses.
In previous research work, this research team has obtained the tryptic recombinant yeast pichia pastoris engineering bacteria (CN 102094040A) using a-factor as secreting signal peptide.
The present invention, by constructing the trypsinase containing exocytosis signal peptide, achieves trypsinase pichia spp efficient secretory expression, solves the problem that trypsinase yields poorly.May be because the pre-region sequence of α-facotor signal peptide plays an important role in heterologous protein exocytosis process.α-factor comprises pre-region, pro-region two portions, and the pre-region sequence of α-factor is consistent with the sequence of α mf signal peptide, Pre-region sequence guides heterologous protein to be positioned at from kytoplasm in endoplasmic reticulum, and the form of heterologous protein with exocytosis after this completes posttranslational modification is secreted into outside born of the same parents.And α-factor signal peptide needs two step secretion process in guiding heterologous protein exocytosis process: 1) pre-region sequence cutting; 2) pro-region sequence cutting.Amalgamation and expression α mf signal peptide is compared with α-factor signal peptide, and cutting efficiency is higher.The recombination engineering bacteria that application the inventive method obtains produces trypsinase, and output is high, is convenient to suitability for industrialized production.The present invention is that the outer efficient secretion trypsinase research work of pichia spp born of the same parents is laid a good foundation.
Accompanying drawing explanation
Fig. 1: cloning vector design of graphics
Fig. 2: the trypsinase enzyme of recombinant yeast pichia pastoris engineering bacteria is lived
Fig. 3: α-factor signal peptide sequence is analyzed
Embodiment
Trypsinase enzyme activity determination method: the amido linkage of trypsinase specificity cutting BAPNA (the p-oil of mirbane acid amides of Na-benzoyl-DL-) carboxyl terminal, product p-nitroanilide (having obtained the maximum absorption at 410nm place).At 37 DEG C, measure 100 μ L crude enzyme liquids with 900 μ L 10mM BAPNA solution (being dissolved in 50mM pH8.0 Tris-HCl damping fluid) in the reaction tank of optical path 0.5cm, the light absorption value change under 410nm in 10min.Enzyme is lived and is defined as: at 37 DEG C, and the enzyme amount that per minute 410nm place absorbance value raises required for 0.1 is 1 trypsin hydrolyzing unit.
The structure of embodiment 1 recombinant plasmid
SGT gene (Gen Bank Accession No.M64471) total length 672bp.According to the nucleotide sequence design primer of 8 kinds of signal peptides.Through two-step pcr the gene order shown in signal peptide sequence and SEQ ID NO.9 carried out with and, obtain fusion gene fragment (with trypsase gene shown in SEQ ID NO.9 be template F2, R primer PCR rear pillar reclaims, reclaim product with F1, R primer PCR post again and obtain fusion fragment, the sequence of F1, F2, R primer is respectively as shown in SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12), according to construction process as shown in Figure 1, merge fragment and pPIC9K plasmid with NotI, BamH I double digestion, after purifying, T4 ligase enzyme 16 DEG C spends the night connection.Connect product chemistry method and transform JM109 competent cell.Conversion fluid coating is dull and stereotyped containing kantlex (50mg/L) LB, extracts the recombinant plasmid that plasmid sequence verification builds.Examining order is completed by the raw work in Shanghai.
Embodiment 2 is produced trypsinase Yeast engineering bacteria and is built
By containing the recombinant plasmid Sal I linearizing of recombination that with the addition of signal peptide before trypsase gene, electroporated pichia pastoris GS115 competent cell, concrete grammar is as follows:
1) the pichia pastoris GS115 of YPD flat board activation is inoculated in 25mL/250mL triangular flask, 30 DEG C of incubated overnight; The above-mentioned nutrient solution of 1% inoculation is in 50mL/500mL triangular flask, and cultivating cell concentration OD600 is 1.3 ~ 1.5;
2) 5000r/min, 4 DEG C of centrifugal 10min collect thalline, use 50mL, 25mL sterilized water suspension cell respectively;
3) the resuspended above-mentioned cell of 5mL 1M sorbyl alcohol, 5000r/min, 4 DEG C of centrifugal 10min collect thalline;
4) the resuspended above-mentioned cell of 500 μ L 1M sorbyl alcohol, packing 80 μ L/1.5mL EP manages and is used for Electroporation-competent cells;
5) 20 μ L linearization plasmids mix with above-mentioned 80 μ L competent cells, leave standstill 15min on ice;
6) said mixture adds aseptic electricity conversion cup (0.2cm) of precooling, and 1500V, 25 μ F, 200 Ω electric shocks once, add 1mL 1M sorbyl alcohol;
7) get said mixture 150 μ L and be coated with MD flat board, cultivate 3 days for 30 DEG C;
8) white colony in the above-mentioned flat board of picking, respectively dibbling 1,2,3, in 4mg/mL (Geneticin) YPD flat board, select single bacterium colony in 4mg/mL Geneticin flat board for shake flask fermentation.
The structure of embodiment 3 recombinant bacterium
Adopt the method for two-step pcr similar to Example 1 by signal peptide (the respectively called after α as of nucleotide sequence as SEQ ID NO.1 to SEQ ID NO.8, Sas, Ipr, Iss, Lss, nsB, α mf, Kps), before being fused to the trypsase gene shown in SEQ ID NO.9,8 recombination α as-mt, Sas-mt, Ipr-mt, Iss-mt, Lss-mt, nsB-mt, α mf-mt, Kps-mt are obtained; Described recombination is connected respectively to yeast expression vector, obtains recombinant plasmid; By described recombinant plasmid electricity conversion pichia spp Pichia pastoris GS115 respectively, obtain recombination yeast engineering strain.
Embodiment 4 recombinant yeast pichia pastoris shake-flask culture
Engineering bacteria the present invention built, as production bacterial strain, contains α-factor signal peptide trypsinase recombinant bacterium for contrast with what build in CN 102094040A, in the activation of YPD flat board.Seed liquor is cultivated, and inoculation 50mL/250mL seed culture medium, 30 DEG C, 220r/min cultivates 24h.Centrifugal seed liquor is OD600=1 inoculation fermentation substratum by seed final concentration in fermention medium, and 30 DEG C, 220r/min cultivates 5d.Survey trypsinase trypsinase enzyme every 24h sampling to live.As shown in Figure 2, what the present invention built lives containing there being six kinds to improve trypsinase enzyme all to some extent in 8 kinds of recombinant bacteriums of unlike signal peptide result, and the enzyme of the recombinant bacterium wherein containing α mf signal peptide is lived the highest.Merge the trypsinase of α mf signal peptide compared with α-factor signal peptide (CK), trypsinase enzyme is lived and is improve 2.75 times.Fig. 3 is the sequence of α-factor signal peptide, can see, α-factor comprises pre-region, pro-region two portions, and the pre-region sequence of α-factor is consistent with the sequence of α mf signal peptide.This shows, the pre-region sequence of α-facotor signal peptide plays an important role in heterologous protein exocytosis process.Pre-region sequence guides heterologous protein to be positioned at from kytoplasm in endoplasmic reticulum, and the form of heterologous protein with exocytosis after this completes posttranslational modification is secreted into outside born of the same parents.And α-factor signal peptide needs two step secretion process in guiding heterologous protein exocytosis process: 1) pre-region sequence cutting; 2) pro-region sequence cutting.Amalgamation and expression α mf signal peptide is compared with α-factor signal peptide, and cutting efficiency is higher.
Seed culture medium (g/L): peptone 20, yeast extract 10, glucose 20.
Fermention medium (g/L): K
2hPO
43H
2o1.51; KH
2pO
45.91; Vitamin H 0.2; YNB (yeast is without amino acid nitrogenous source) 13.4; Tryptones 10; Yeast powder 5; Vitamin H 4 × 10
-4; Methyl alcohol 1%.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (9)
1. one kind is produced tryptic recombination microzyme, it is characterized in that, be nucleotide sequence be before the trypsase gene of sequence shown in SEQ ID NO.9 fusion nucleus nucleotide sequence be sequence shown in SEQ ID NO.7 signal peptide formed recombination, recombination is carried out in Host Strains express the recombination microzyme obtained.
2. recombination microzyme according to claim 1, it is characterized in that, the Host Strains that sets out of described recombination microzyme be following any one: pichia pastoris GS115, pichia pastoris KM71, pichia pastoris X-33, pichia pastoris SMD1168.
3. recombination microzyme according to claim 1, is characterized in that, the Host Strains that sets out of described recombination microzyme is pichia pastoris GS115.
4. recombination microzyme according to claim 1, it is characterized in that, described signal peptide by nucleotides sequence be classified as following any one signal peptide substitute: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.8.
5. the construction process of recombination microzyme described in a claim 1, before nucleotide sequence is the trypsase gene of the sequence shown in SEQ ID NO.9, add the signal peptide formation recombination that nucleotide sequence is sequence shown in SEQ ID NO.7, this recombination is connected in expression plasmid and forms recombinant plasmid, then be transformed in host yeast and obtain recombinant bacterium.
6. method according to claim 5, is characterized in that, described expression plasmid be following any one: pGAP ZA, pAO815, pGAP α A, pPIC9K, pPIC ZB.
7. method according to claim 5, is characterized in that, described expression plasmid pPIC9K.
8. method according to claim 5, is characterized in that, described construction process for: (1) composition sequence is the gene fragment of sequence shown in SEQ ID NO.9; (2) with the gene fragment of step 1 for template, use sequence is SEQ ID NO.11, the primer shown in SEQ ID NO.12 carries out PCR, as template after PCR primer reclaims, with sequence be SEQ ID NO.10, the primer shown in SEQ ID NO.12 carries out PCR and obtains recombination; (3) recombination obtained by NotI, BamHI double digestion step 2 and pPIC9K plasmid, connect with ligase enzyme and obtain recombinant plasmid; (4) recombinant plasmid transformed step 3 obtained, in pichia pastoris GS115, namely obtains recombinant yeast pichia pastoris bacterium.
9. the application of recombination microzyme described in claim 1 in trypsinase production.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893700A (en) * | 2017-04-19 | 2017-06-27 | 江南大学 | A kind of method that engineer's self-activation leader peptide sequences improve trypsase enzyme activity |
| CN110144319A (en) * | 2019-04-24 | 2019-08-20 | 天津科技大学 | The genetic engineering bacterium and its construction method of efficient heterogenous expression alkali protease |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000017332A1 (en) * | 1998-09-21 | 2000-03-30 | Eli Lilly And Company | Production of soluble recombinant trypsinogen analogs |
| CN103173367A (en) * | 2013-03-11 | 2013-06-26 | 江南大学 | Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application |
| WO2014067926A1 (en) * | 2012-10-29 | 2014-05-08 | Lonza Ltd | Expression sequences |
-
2014
- 2014-10-17 CN CN201410555749.2A patent/CN104312933B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000017332A1 (en) * | 1998-09-21 | 2000-03-30 | Eli Lilly And Company | Production of soluble recombinant trypsinogen analogs |
| WO2014067926A1 (en) * | 2012-10-29 | 2014-05-08 | Lonza Ltd | Expression sequences |
| CN103173367A (en) * | 2013-03-11 | 2013-06-26 | 江南大学 | Engineered Saccharomyces cerevisiae producing heat-stability recombinant trypsin, and its application |
Non-Patent Citations (5)
| Title |
|---|
| ANDREAS KÜBERL ET AL.: "High-quality genome sequence of Pichia pastoris CBS7435", 《JOURNAL OF BIOTECHNOLOGY》 * |
| GENBANK: "GenBank：EU285586.1", 《GENBANK》 * |
| GENBANK: "GenBank：M64471.1", 《GENBANK》 * |
| LEONARDO M. DAMASCENO ET AL.: "Protein secretion in Pichia pastoris and advances in protein production", 《APPL MICROBIOL BIOTECHNOL》 * |
| ZHENMIN LING ET AL.: "Rational design of a novel propeptide for improving active production of Streptomyces griseus trypsin in Pichia pastoris", 《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106893700A (en) * | 2017-04-19 | 2017-06-27 | 江南大学 | A kind of method that engineer's self-activation leader peptide sequences improve trypsase enzyme activity |
| CN106893700B (en) * | 2017-04-19 | 2020-07-07 | 江南大学 | Method for improving trypsin enzyme activity by artificially designing self-activating leader peptide sequence |
| CN110144319A (en) * | 2019-04-24 | 2019-08-20 | 天津科技大学 | The genetic engineering bacterium and its construction method of efficient heterogenous expression alkali protease |
| CN110144319B (en) * | 2019-04-24 | 2021-01-15 | 天津科技大学 | Gene engineering bacterium for high-efficiency heterologous expression of alkaline protease and construction method thereof |
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