CN108795964A - Recombinate production method of the PRESCISSION enzymes in Escherichia coli - Google Patents
Recombinate production method of the PRESCISSION enzymes in Escherichia coli Download PDFInfo
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Abstract
The invention discloses production method of the recombination PRESCISSION enzymes in Escherichia coli, include the following steps：The structure of S1, PGEX-4T-1-PRESCISSION protease expression plasmid；The induced expression of S2, PRESCISSION protease and identification；The purifying and identification of S3, PRESCISSION protease.Present invention application escherichia expression system, by designing expression vector, realize recombination PRESCISSION protease in expression in escherichia coli, target protein enzyme is purified using the methods of affinity chromatography, 75 gel permeation chromatographies of Superdex, the purifying flow and purification time of target protein enzyme is greatly shortened, the purity, yield and enzyme activity of protease are improved simultaneously, save experimental procedure and reduce cost.The method of the present invention is laid a good foundation to research and develop and producing other protide toolenzymes.
Description
Technical field
The present invention relates to polymer reaction fields, specially recombinate producer of the PRESCISSION enzymes in Escherichia coli
Method.
Technical background
Currently, protein yield and activity it is not high be heterologous protein expression especially toxic protein expression technical bottleneck.With
The development of biotechnology, researcher is using amalgamation and expression policies toxicity and the expression difficulty of slightly solubility albumen.
PRESCISSION protease has height digestion specificity, recognition site Leu-Glu-Val-Leu-Phe-
Gln Gly-Pro, can cut to idiocrasy peptide bond between Gln-Gly, PRESCISSION protease overall lengths 546bp or so,
The protein relative molecular mass of coding is about 19997.Given this specific, this research builds weight by gene engineering method
Group PRESCISSION protease is to develop a kind of novel toolenzyme.ERC group virus is searched using ncbi database
The amino acid sequence of PRESCISSION protease is optimized for by biological tool website suitable for Bacillus coli expression
Gene order is directly synthesized and is integrated into expression vector, is expressed and is purified the PRESCISSION protease for obtaining high-purity.
The fusion protein of the protease cutting site containing PRESCISSION is cut to the protease energy idiocrasy, there is good biology
Activity, the genetic engineering tools enzyme to develop new are had laid a good foundation.
The fusion protein in the site containing PRESCISSION is cut to PRESCISSION protease energy idiocrasys,
PRESCISSION protease cutting sites are commonly used for the excision of client's fusion protein label, high-purity and high activity
Succeeding in developing for PRESCISSION protease will generate certain commercial value.
Currently, PRESCISSION protease in the market, bioactivity is generally relatively low, and common PRESCISSION
Production method of protease, complex steps, cost are higher.Therefore, a kind of recombination PRESCISSION enzymes of research and development are in Escherichia coli
Production method, realize high-purity recombination PRESCISSION protease preparation, to save experimental procedure and reduce cost,
It is necessary.
Invention content
It is an object of the invention to：Production method of the recombination PRESCISSION enzymes in Escherichia coli is provided, to solve
Above-mentioned technical problem.
To achieve the goals above, the present invention provides the following technical solutions：
Production method of the PRESCISSION enzymes in Escherichia coli is recombinated, is included the following steps：
The structure of S1, PGEX-4T-1-PRESCISSION protease expression plasmid；
The induced expression of S2, PRESCISSION protease and identification；
The purifying and identification of S3, PRESCISSION protease.
Preferably, the step S1, specifically divides following steps：
A, according to the gene order after codon optimization, PRESCISSION protease directly carries out gene chemical synthesis；
B, PGEX-4T-1 vector plasmids are linearized by digestion, linearisation product is carried out with gene chemical synthesis product
Seamless structure forms recombinant products；
C, it takes 20ul recombinant products to be transferred in TOP10 competent cells, stands 30min on ice, then 42 DEG C of heat shocks
90s, then stand 2min on ice, adds 600ul LB liquid mediums, 37 DEG C, shaking table culture 45min under the conditions of 200rpm,
It is coated in the LB tablets of the ampicillin containing 50ug/mL, is finally putting into 37 DEG C of incubator overnight incubations；
D, using sterilizing toothpick from LB tablets random four bacterium colonies of picking, access LBs of the 4ml containing amicillin resistance
In culture medium；37 DEG C, shaking table culture is stayed overnight under the conditions of 200rpm, extracting plasmid and the sequencing of Sanger methods.
Preferably, the step S2, specifically divides following steps：
A, Sanger method sequencing results will be passed through in step S1 and is accredited as positive PGEX-4T-1-PRESCISSION eggs
White enzyme plasmid is transferred in competence BL21 (DE3), is coated on the LB tablets containing amicillin resistance, and 37 DEG C are inverted culture
Overnight；
B, it is placed in the LB culture mediums containing amicillin resistance using single bacterium colony on sterilizing toothpick picking LB tablets, 37
DEG C, overnight incubation under the conditions of 220rpm, with volume ratio 1:100 ratio connects bacterium solution in LB culture mediums of the 4ml containing ampicillin
In, continue to cultivate 2.5h, IPTG to 0.5mM/L is added when reaching 0.6~0.8 in OD600nm, in 15 DEG C of overnight inducible protein tables
It reaches；
C, the 2ml bacterium solutions for taking induced expression centrifuge 1min collection thalline under the conditions of 12000rpm, the 1 of 100ul are added
× protein electrophoresis buffer solution boils 5min, centrifuges 1min under the conditions of 12000rpm after cooling, take 10ul thalline electrophoretic buffers
Sample carries out 12% polyacrylamide gel electrophoresis, removes gel and utilizes quick dyeing instrument dyeing-decolorzing 10min, observation induction
As a result the variation of front and back protein band is shown in the thalline after induced expression and occurs and PRESCISSION protease theories point
Son measures consistent band of expression, and do not have band presence in the thalline before inducing, it was demonstrated that PRESCISSION protease tables
Up to normal.
Preferably, the step S3, specifically divides following steps：
A, the BL21 for being transferred to PGEX-4T-1-PRESCISSION protease plasmids will be incubated overnight in S2-a steps
(DE3) bacterium solution is with volume ratio 1:100 ratio is connected in 800ml LB containing ampicillin, 37 DEG C, cultivate under the conditions of 220rpm
When 2h or so, OD600nm reach 0.6-0.8, IPTG to final concentration 0.5mM/L is added, is expressed in 15 DEG C of overnight inducible proteins,
8000rpm centrifuges 10min, collects thalline, -20 DEG C of preservations；
B, will -20 DEG C freeze thalline and cell pyrolysis liquid be added (ratio is：1g thalline correspond to 5mL cell pyrolysis liquids), it uses
Sonicator, ultrasound 3 seconds under cryogenic conditions stop 10 seconds, run 10 minutes carry out brokenly bacterium, 4 DEG C, 16000rpm conditions altogether
Lower centrifugation 15min, separation supernatant and precipitation；
C, supernatant, precipitation are taken respectively, and is detected using SDS-PAGE methods, and testing result is broken rear supernatant
There is the band consistent with PRESCISSION protease theoretical molecular weights in precipitation, contain 80% or more protease in supernatant,
Illustrate that broken results are good；
D, the supernatant that will be detached in S3-b steps is placed in the centrifuge tube of clean 50ml and 2ml GST columns is added, 4 DEG C
It is incubated 2h, is first cleaned with cell pyrolysis liquid 100ml, then with the cell pyrolysis liquid containing 20mM/L reduced glutathiones
5ml is eluted, finally by eluent using PRESCISSION protease storing liquid as mobile phase over-molecular sieve Superdex
75, the eluent of destination protein is collected according to 280nm ultraviolet absorption values, and destination protein liquid is carried out with 12% SDS-PAGE
Detection, to which the PRESCISSION protease of purifying be made.
Preferably, in step S3-b, S3-d, the trihydroxy methyl amino first containing 20mM/L in the cell pyrolysis liquid
Alkane, the sodium chloride of 300mM/L, the glycerine that weight percent is 10%, the cell pyrolysis liquid PH are 8.0.
The beneficial effects of the present invention are：
Present invention application escherichia expression system realizes recombination PRESCISSION albumen by designing expression vector
Enzyme is in expression in escherichia coli, using affinity chromatography and 75 gel permeation chromatographies of Superdex to PRESCISSION albumen
Enzyme is purified, and whole process of purification only needs 5~6h, simple two-step purifying mode to be integrally greatly shortened
The purifying flow and purification time of PRESCISSION protease, while improving the purity of PRESCISSION protease, yield
And enzyme activity, and merge have GST labels PRESCISSION protease can to the target protein with the sites PRESCISSION into
It is purified after row digestion, GST column effluxes can be obtained concentration and the higher native protein of purity after collecting digestion, with others
Digestion way of purification is compared to saving experimental procedure and reduces cost.The new method of the PRESCISSION protease purification techniques,
It lays a good foundation to research and develop and producing other protide toolenzymes.
Specific implementation mode
In order to facilitate the understanding of those skilled in the art, with reference to specific embodiment, the present invention is further illustrated.
Embodiment 1：
Production method of the PRESCISSION enzymes in Escherichia coli is recombinated, is included the following steps：
The structure of S1, PGEX-4T-1-PRESCISSION protease expression plasmid, specifically divides following steps：
A, according to the gene order after codon optimization, PRESCISSION protease directly carries out gene chemical synthesis；
B, PGEX-4T-1 vector plasmids are linearized by digestion, linearisation product is carried out with gene chemical synthesis product
Seamless structure forms recombinant products；
C, it takes 20ul recombinant products to be transferred in TOP10 competent cells, stands 30min on ice, then 42 DEG C of heat shocks
90s, then stand 2min on ice, adds 600ul LB liquid mediums, 37 DEG C, shaking table culture 45min under the conditions of 200rpm,
It is coated in the LB tablets of the ampicillin containing 50ug/mL, is finally putting into 37 DEG C of incubator overnight incubations；
D, using sterilizing toothpick from LB tablets random four bacterium colonies of picking, access LBs of the 4ml containing amicillin resistance
In culture medium；37 DEG C, shaking table culture is stayed overnight under the conditions of 200rpm, extracting plasmid and the sequencing of Sanger methods.
The induced expression of S2, PRESCISSION protease and identification, specifically divide following steps：
A, Sanger method sequencing results will be passed through in step S1 and is accredited as positive PGEX-4T-1-PRESCISSION eggs
White enzyme plasmid is transferred in competence BL21 (DE3), is coated on the LB tablets containing amicillin resistance, and 37 DEG C are inverted culture
Overnight；
B, it is placed in the LB culture mediums containing amicillin resistance using single bacterium colony on sterilizing toothpick picking LB tablets, 37
DEG C, overnight incubation under the conditions of 220rpm, with volume ratio 1:100 ratio connects bacterium solution in LB culture mediums of the 4ml containing ampicillin
In, continue to cultivate 2.5h, IPTG to 0.5mM/L is added when reaching 0.6~0.8 in OD600nm, in 15 DEG C of overnight inducible protein tables
It reaches；
C, the 2ml bacterium solutions for taking induced expression centrifuge 1min collection thalline under the conditions of 12000rpm, the 1 of 100ul are added
× protein electrophoresis buffer solution boils 5min, centrifuges 1min under the conditions of 12000rpm after cooling, take 10ul thalline electrophoretic buffers
Sample carries out 12% polyacrylamide gel electrophoresis, removes gel and utilizes quick dyeing instrument dyeing-decolorzing 10min, observation induction
As a result the variation of front and back protein band is shown in the thalline after induced expression and occurs and PRESCISSION protease theories point
Son measures consistent band of expression, and do not have band presence in the thalline before inducing, it was demonstrated that PRESCISSION protease tables
Up to normal.
The purifying and identification of S3, PRESCISSION protease, specifically divide following steps：
A, the BL21 for being transferred to PGEX-4T-1-PRESCISSION protease plasmids will be incubated overnight in S2-a steps
(DE3) bacterium solution is with volume ratio 1:100 ratio is connected in 800ml LB containing ampicillin, 37 DEG C, cultivate under the conditions of 220rpm
When 2h or so, OD600nm reach 0.6-0.8, IPTG to final concentration 0.5mM/L is added, is expressed in 15 DEG C of overnight inducible proteins,
8000rpm centrifuges 10min, collects thalline, -20 DEG C of preservations；
B, will -20 DEG C freeze thalline and cell pyrolysis liquid be added (ratio is：1g thalline correspond to 5mL cell pyrolysis liquids), it uses
Sonicator, ultrasound 3 seconds under cryogenic conditions stop 10 seconds, run 10 minutes carry out brokenly bacterium, 4 DEG C, 16000rpm conditions altogether
Lower centrifugation 15min, separation supernatant and precipitation；
C, supernatant, precipitation are taken respectively, and is detected using SDS-PAGE methods, and testing result is broken rear supernatant
There is the band consistent with PRESCISSION protease theoretical molecular weights in precipitation, contain 80% or more protease in supernatant,
Illustrate that broken results are good；
D, the supernatant that will be detached in S3-b steps is placed in the centrifuge tube of clean 50ml and 2ml GST columns is added, 4 DEG C
It is incubated 2h, is first cleaned with cell pyrolysis liquid 100ml, then with the cell pyrolysis liquid containing 20mM/L reduced glutathiones
5ml is eluted, finally by eluent using PRESCISSION protease storing liquid as mobile phase over-molecular sieve Superdex
75, the eluent of destination protein is collected according to 280nm ultraviolet absorption values, and destination protein liquid is carried out with 12% SDS-PAGE
Detection, to which the PRESCISSION protease of purifying be made.
In step S3-b, S3-d, the trishydroxymethylaminomethane containing 20mM/L in the cell pyrolysis liquid,
The sodium chloride of 300mM/L, the glycerine that weight percent is 10%, the cell pyrolysis liquid PH are 8.0.
It is above-mentioned that invention is exemplarily described, it is clear that present invention specific implementation is not subject to the restrictions described above, only
Use this insubstantial improvement of inventive concept and technical scheme of the present invention progress, or the not improved structure by invention
Think and technical solution directly applies to other occasions, within protection scope of the present invention.
Claims (5)
1. recombinating production method of the PRESCISSION enzymes in Escherichia coli, which is characterized in that include the following steps：
The structure of S1, PGEX-4T-1-PRESCISSION protease expression plasmid；
The induced expression of S2, PRESCISSION protease and identification；
The purifying and identification of S3, PRESCISSION protease.
2. production method of the recombination PRESCISSION enzymes according to claim 1 in Escherichia coli, which is characterized in that
The step S1, specifically divides following steps：
A, according to the gene order after codon optimization, PRESCISSION protease directly carries out gene chemical synthesis；
B, PGEX-4T-1 vector plasmids are linearized by digestion, linearisation product carries out seamless with gene chemical synthesis product
Structure forms recombinant products；
C, it takes 20ul recombinant products to be transferred in TOP10 competent cells, stands 30min on ice, then 42 DEG C of heat shock 90s, then
2min is stood on ice, adds 600ul LB liquid mediums, and 37 DEG C, shaking table culture 45min under the conditions of 200rpm are coated on and contain
In the LB tablets of 50ug/mL ampicillins, it is finally putting into 37 DEG C of incubator overnight incubations；
D, random four bacterium colonies of picking, LB cultures of the access 4ml containing amicillin resistance from LB tablets using sterilizing toothpick
In base；37 DEG C, shaking table culture is stayed overnight under the conditions of 200rpm, extracting plasmid and the sequencing of Sanger methods.
3. production method of the recombination PRESCISSION enzymes according to claim 2 in Escherichia coli, which is characterized in that
The step S2, specifically divides following steps：
A, Sanger method sequencing results will be passed through in step S1 and is accredited as positive PGEX-4T-1-PRESCISSION protease matter
Grain is transferred in competence BL21 (DE3), is coated on the LB tablets containing amicillin resistance, 37 DEG C of inversion overnight incubations；
B, it is placed in the LB culture mediums containing amicillin resistance using single bacterium colony on sterilizing toothpick picking LB tablets, 37 DEG C,
Overnight incubation under the conditions of 220rpm, with volume ratio 1:100 ratio connects bacterium solution in LB culture mediums of the 4ml containing ampicillin,
Continue to cultivate 2.5h, IPTG to 0.5mM/L is added when reaching 0.6~0.8 in OD600nm, in 15 DEG C of overnight inducible protein expression；
C, the 2ml bacterium solutions for taking induced expression centrifuge 1min collection thalline under the conditions of 12000rpm, 1 × albumen of 100ul are added
Electrophoretic buffer boils 5min, 1min is centrifuged under the conditions of 12000rpm after cooling, 10ul thalline electrophoretic buffer samples is taken to carry out
12% polyacrylamide gel electrophoresis removes gel and utilizes quick dyeing instrument dyeing-decolorzing 10min, the front and back albumen one of observation induction
As a result it is consistent with PRESCISSION protease theoretical molecular weights to be shown in appearance in the thalline after induced expression for the variation of band
Band of expression, and there is no band presence in the thalline before inducing, it was demonstrated that PRESCISSION albumen expression of enzymes is normal.
4. production method of the recombination PRESCISSION enzymes according to claim 3 in Escherichia coli, which is characterized in that
The step S3, specifically divides following steps：
A, BL21 (DE3) bacterium for being transferred to PGEX-4T-1-PRESCISSION protease plasmids will be incubated overnight in S2-a steps
Liquid is with volume ratio 1:100 ratio is connected in 800ml LB containing ampicillin, 37 DEG C, culture 2h or so under the conditions of 220rpm,
When OD600nm reaches 0.6-0.8, IPTG to final concentration 0.5mM/L is added, in 15 DEG C of overnight inducible proteins expression, 8000rpm from
Heart 10min collects thalline, -20 DEG C of preservations；
B, will -20 DEG C freeze thalline and cell pyrolysis liquid be added (ratio is：1g thalline correspond to 5mL cell pyrolysis liquids), use ultrasound
Wave is crushed instrument, ultrasound 3 seconds under cryogenic conditions, stops 10 seconds, runs carry out brokenly within 10 minutes bacterium altogether, 4 DEG C, centrifuge under the conditions of 16000rpm
15min, separation supernatant and precipitation；
C, supernatant, precipitation are taken respectively, and is detected using SDS-PAGE methods, and testing result is in broken rear supernatant precipitation
There is the band consistent with PRESCISSION protease theoretical molecular weights, contain 80% or more protease in supernatant, illustrates broken
As a result good；
D, the supernatant that will be detached in S3-b steps is placed in addition 2ml GST columns in the centrifuge tube of clean 50ml, 4 DEG C of incubations
2h is first cleaned with cell pyrolysis liquid 100ml, then with the cell pyrolysis liquid 5ml containing 20mM/L reduced glutathiones into
Row elution, finally by eluent using PRESCISSION protease storing liquid as mobile phase over-molecular sieve Superdex 75, root
The eluent that destination protein is collected according to 280nm ultraviolet absorption values, destination protein liquid is detected with 12% SDS-PAGE, from
And the PRESCISSION protease of purifying is made.
5. production method of the recombination PRESCISSION enzymes according to claim 4 in Escherichia coli, which is characterized in that
In step S3-b, S3-d, the chlorination of the trishydroxymethylaminomethane containing 20mM/L, 300mM/L in the cell pyrolysis liquid
Sodium, the glycerine that weight percent is 10%, the cell pyrolysis liquid PH are 8.0.
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| US20050106671A1 (en) * | 2003-11-18 | 2005-05-19 | Darcy Birse | Expression vector, host cell and method for producing fusion proteins |
| CN106692961A (en) * | 2015-11-17 | 2017-05-24 | 长春市福迪奥美科技有限公司 | Arginase composition, arginase activator and application thereof |
| WO2017162297A1 (en) * | 2016-03-24 | 2017-09-28 | Curevac Ag | Immobilized inorganic pyrophosphatase (ppase) |
| CN107267545A (en) * | 2017-07-26 | 2017-10-20 | 通用生物***（安徽）有限公司 | Recombinate the preparation method of Bsa I restriction enzymes |
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| CB02 | Change of applicant information | ||
| CB02 | Change of applicant information |
Address after: 239000 No. 69, qifushi West Road, Chuzhou economic and Technological Development Zone, Anhui Province Applicant after: General Biology (Anhui) Co.,Ltd. Address before: 239000 No. 6, yongyang Road, Chuzhou economic and Technological Development Zone, Anhui Province Applicant before: GENERAL BIOSYSTEMS (ANHUI), Inc. |
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| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20181113 |