CN103275917A - TEV protease expression engineering bacteria and its construction and application - Google Patents
TEV protease expression engineering bacteria and its construction and application Download PDFInfo
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Abstract
The invention relates to a genome-based TEV protease expression system. The system is obtained in the following way: a TEV protease gene which is actuated by a T7 strong promoter and is fused with a maltose-binding protein gene malE and a chloramphenicol resistant gene are integrated together into a escherichia coli expression bacterial strain BL21(DE3) genome by a homologous recombination method mediated by a recombinase from lambda bacteriophage, and replace a malE gene area in the genome. An escherichia coli expression bacterial strain CGMCC No. 7482 is adopted. The TEV protease can be purified by one-step affinity chromatography, and the yield can reach 4.2mg/L. Separated and purified TEV protease has a good digestion activity. The TEV protease expression system can be a conventional means for expression and purification of the TEV protease.
Description
Technical field
The present invention relates to the genetically engineered field, specifically relate to a kind of based on genomic TEV proteolytic enzyme expression system.
Background technology
Heterologous protein carries out the routine operation that great expression is biological chemistry and molecular biology research in intestinal bacteria, also be the important means of research gene and protein function.Heterologous protein is because wide material sources, its employed codon usually has different significantly with intestinal bacteria, thereby can not secrete to intercellular substance, namely can not only can show with the form of inclusion body with the form of soluble proteins, this point is particularly evident when eukaryotic gene is expressed.Address this problem one commonly used and effectively means be that goal gene and fusion tag are carried out amalgamation and expression.Fusion tag commonly used has maltose binding protein (MBP), ubiquitin sample modified protein (SUMO), nitrogenous source to utilize substance A (NusA) and Thiadiazolidine isomerase (GST) etc.Solubility when fusion tag not only can increase the target protein heterogenous expression also might increase its output.The restriction enzyme site of design proteolytic enzyme of being everlasting between fusion tag and the target protein makes the mode that can cut by enzyme with the fusion tag of purifying and target protein separately.Common proteolytic enzyme comprises TEV proteolytic enzyme, zymoplasm, enteropeptidase etc., and is wherein important with TEV proteolytic enzyme.
TEV proteolytic enzyme is that the Nla(nuclear that derives from marmor erodens comprises protein gene) a kind of proteolytic enzyme.The aminoacid sequence of TEV effect is ENLYFQ ↓ G (↓ expression restriction enzyme site), because TEV proteolytic enzyme has good enzyme to cut the tolerance of specificity and enzyme tangent condition (namely all showing good active under multiple condition), therefore become one of proteolytic enzyme the most widely of present utilization, its purposes mainly comprises: in the separation of fusion rotein and the purifying of target protein, genomics and the proteomics research mark of specific protein with separate etc.
The method that obtains at present TEV proteolytic enzyme be with its gene clone in expression vector, in the gained recombinant plasmid transformed e. coli host bacteria after, carry out separation and purification behind the abduction delivering TEV proteolytic enzyme again.Although this expression system based on plasmid is widely adopted good effective, exist some intrinsic deficiencies.As the conversion of need plasmid, need to use microbiotic (antibiotic use increased the difficulty of separation and purification of enzyme and the price of finished product), clone in the gene on the plasmid and exist the activity of bacterial strain heterogeneity and gene after long-time the use, can reduce etc. problem.These all might be to expression of gene and enzyme active unfavorable.
Summary of the invention
At the problems referred to above, the invention provides a kind of based on genomic TEV proteolytic enzyme expression system.
The employed escherichia coli expression bacterial strain of this expression system is colon bacillus (Escherichia coli) CGMCC No.7482.
TEV proteolytic enzyme expression system is to be obtained by following method: at first TEV proteinase gene and maltose binding protein gene malE merged, and the base sequence of design TEV proteolytic enzyme restriction enzyme site between two genes, fusion gene is driven by the T7 strong promoter.Fusion gene is cloned in chloramphenicol resistance gene again, and the homologous recombination method that the homologous fragment of this box gene by malE gene upstream and downstream both sides mediated by the recombinase that derives from lambda particles phage is integrated into the malE zone of above-mentioned bacterial strains at last.
The employed recombinase that derives from lambda particles phage of present patent application uses red α, these three manipulators that gene is formed of red β and red γ, red α genes encoding 5' → 3'DNA excision enzyme, red β genes encoding single strand binding protein, also exercise the activity of recombinase, red γ genes encoding Gam albumen can stop the excision enzyme of thalline inside to the degraded of foreign DNA.The recombinase gene manipulator place by the LacI gene under the pLac promotor of rigorous adjusting, pLac expresses under isopropyl-(IPTG) is induced.Homologous recombination between the recombinase catalysis homologous fragment is referred to as recombined engineering, and the recombined engineering method is easy and efficient.
The invention also discloses above-mentioned TEV proteolytic enzyme and express the application of engineering bacteria in expressing TEV proteolytic enzyme.Experimental results show that malE-TEV fusion gene under the T7 strong promoter control in the CGMCC No.7482 genome is under IPTG induces, the MBP-TEV amalgamation and expression also acts on the TEV proteolytic enzyme restriction enzyme site of fusion rotein self, obtain and the TEV proteolytic enzyme that separates by the shearing in somatic cells, this has just been avoided separating at external purifying MBP-TEV numerous and diverse step of TEV again.Efficiently express TEV proteolytic enzyme output and can reach 4.2mg/L.Enzyme is cut the fusion rotein experiment and is shown that the TEV proteolytic enzyme of separation has good enzyme and cuts activity.
Adopt colon bacillus CGMCC No.7482 bacterial strain, be based on the advantage of genomic TEV proteolytic enzyme expression system: avoided a series of problems based on pUC pUC, simple and efficient to handle, therefore the output height may become the means of good expression and purifying TEV proteolytic enzyme.
TEV is utilization scope proteolytic enzyme very widely, and at expression and the purifying of albumen, the protein labeling in genomics and the proteomics research and processing all are commonly employed.Present TEV is the product of commercialization major company, and is expensive, is 2063 yuan of per 200 units as the price of the AcTEV of American I nvitrogen company, can infer thus, and expressed and TEV purifying of body series will have excellent economic value.
Description of drawings
Fig. 1 is the inventive method synoptic diagram.
Fig. 2 is the gel electrophoresis result of each strain gene type checking of experimentation of the present invention.
1.DL2000DNA molecular weight standard: 2.0,1.0,0.75,0.5,0.25,0.1kb
2.BL21 (DE3) genome is template, the 2.4kb product of R1073-R1074PCR amplification
3.LS2401 genome is template, the 1.9kb product of R1073-R1075PCR amplification
4.LS2401 genome is template, the 1.5kb product of R1061-R1074PCR amplification
5.LS2401 genome is template, the 4.1kb product of R1073-R1074PCR amplification
6. λ/HindIII molecular weight standard: 23.1,9.4,6.6,4.4,2.3,2.0kb.
Fig. 3 is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result that engineering strain LS2401 expressed proteins and Ni-NTA purifying after IPTG induces obtain TEV.
1. protein molecular weight standard: 116.0,66.2,45.0,35.0,25.0,18.4,14.4KD;
2.LS2401 the total protein of expressing, MBP is 41.0KD, and His-TEV is 28.6KD;
3.LS2401 the soluble proteins of expressing, MBP is 41.0KD, and His-TEV is 28.6KD;
4.Ni-NTA purifying obtains TEV.
Fig. 4 is the result that TEV proteolytic enzyme enzyme is cut the His-MBP-hRnBP fusion rotein.
1. protein molecular weight standard: 116.0,66.2,45.0,35.0,25.0KD;
2.BL21 (DE3)/and total protein that pLS136 expresses, His-MBP-hRnBP fusion rotein 89.8KD;
3.BL21 (DE3)/and soluble proteins that pLS136 expresses, His-MBP-hRnBP fusion rotein 89.8KD;
4.Ni-NTA purifying obtains His-MBP-hRnBP fusion rotein 89.8KD;
5.TEV cutting, enzyme obtains hRnBP48.1KD, His-MBP41.7KD.
Fig. 5 is the result that TEV proteolytic enzyme enzyme is cut the His-TIG-PMT0106 fusion rotein.
1. protein molecular weight standard: 116.0,66.2,45.0,35.0,25.0,18.4,14.4KD;
2.BL21 (DE3)/and total protein that pLS138 expresses, His-TIG-PMT0106 fusion rotein 88.9KD;
3.BL21 (DE3)/and soluble proteins that pLS136 expresses, His-TIG-PMT0106 fusion rotein 88.9KD;
4.Ni-NTA purifying obtains His-TIG-PMT0106 fusion rotein 88.9KD;
5.TEV cutting, enzyme obtains His-TIG albumen 49.9KD, PMT0106 protein 39 .0KD.
Embodiment
Employed term unless other explanation is arranged, generally has the implication of those of ordinary skills' common sense in the present invention.
Below in conjunction with specific embodiment, and comparable data is described the present invention in further detail.Should be understood that these embodiment just in order to demonstrate the invention, but not limit the scope of the invention by any way.
In following embodiment, the various processes of Xiang Ximiaoshuing and method are not ordinary methods as known in the art.The source of agents useful for same, trade(brand)name and be necessary to list its moiety person are all indicated when occurring first, thereafter used identical reagent if no special instructions, all identical with the content of indicating first.
Used bacterial strain and plasmid among the embodiment are disclosed bacterial strain and plasmid:
1.Escherichia?coli?MG1655。Genotype F
-LAM-rph-1 is from U.S. intestinal bacteria preservation center.
2.Escherichia?coli?DH10B。Genotype F
-McrA Δ (mrr-hsdRMS-mcrBC) φ 80 Δ lacZ Δ M15 Δ lacX74deoRrecA1araD139 Δ (ara, leu) 7697galU galK rpsL endA1nupG..Document: Life Technologies, Inc.Focus, 1990,12:19.Available from American I nvitrogen company.
3.Escherichia?coli?BW25141。The coli strain that contains the Pir gene is the host bacterium of the plasmid that contains the R6K replicon.
Document: Datsenko KA, Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.Proc Natl Acad Sci U S A.2000,97 (12): 6640-5.Teach from the Barry Wanner of U.S. Purdue university.
4.Escherichia?coli?BL21(DE3)。Genotype B F
–Dcm ompT hsdS (rB
–MB
–) gal λ (DE3).Available from American I nvitrogen company.
5.pBluescript?II(KS-)。Gene clone carrier is available from U.S. Stratagene company.
6.pET30a(+)。Expression vector is available from U.S. Novagen company.
7.pKD4。Document: Datsenko KA, Wanner BL.One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.Proc Natl Acad Sci U S A.2000,97 (12): 6640-5.Teach from the Barry Wanner of U.S. Purdue university.
8.pMOD4RT-G。Document: Zhang Y, Lindsey Nash L, Fisher A.A simplified, robust,
and?streamlined?procedure?for?the?production?of?C.elegans?transgenes?via
recombineering.BMC?Developmental?Biology2008,8:119。From the U.S.
The Alfred Fisher professor of University of Pittsburgh.
9.pBAD322C。Document: Cronan JE.A family of arabinose-inducible Escherichia coli expression vectors having pBR322copy control.Plasmid.2006,55 (2): 152-7.Teach from the Urbana-Champaign branch school John Cronan of American I llinois university.
10.pTKRed。Document: Kuhlman TE, Cox EC.Site-specific chromosomal integration of large synthetic constructs.Nucleic Acids Research, 2010,38 (6), e92.Teach from the Thomas Kuhlman of U.S. Princeton university.
The structure of embodiment 1. fusion rotein recombinant clones
Design primer MBP1:5'-GAA
GAATTCCATATGCACCATCATCATCATCACGAA
GAAGGTAAACTGGTAATC-3', (SEQ ID NO.1), EcoRI and NdeI restriction enzyme site are represented with underscore; MBP2:5'-GAA
GGATCCCATGGCGCCCTGAAAATAAAGATTCTCAG
TCTGCGCGTCTTTCAGGG-3', (SEQ ID NO.2), BamHI and NcoI restriction enzyme site represent that with underscore the reverse complementary sequence of the base sequence of 7 amino acid ENLYFQG of coding TEV restriction enzyme site is represented with italic.Be template with intestinal bacteria MG1655 genomic dna, the MBP1-MBP2PCR amplification obtains the malE gene fragment of 1.2kb, 1.2kb after cutting with the EcoRI-BamHI enzyme, link to each other with the carrier pKS (-) that same enzyme is cut, the electric transformed competence colibacillus cell that connects product transformed into escherichia coli DH10B is (unless dated especially, below Ke Long host bacterium is DH10B), under the amicillin resistance of 100 μ g/ml, screening obtains recombinant clone pLS406.
Design primer GPM1:5'-GAA
GGATCCCGCCTCCTTCCCGGTCATGCG-3', (SEQ ID NO.3), the BamHI restriction enzyme site is represented with underscore; GPM2:5'-GAA
AAGCTTACCCCC
GGCCAGGCCAACTAC-3', (SEQ ID NO.4), the HindIII restriction enzyme site is represented with underscore.Genomic dna with streptomyces hygroscopicus Streptomyces hygroscopicus var.geldanus (from U.S. representative microbial DSMZ) is masterplate, GPM1 and GPM2PCR amplification obtain 1.5kb geldanamycin biological synthesis gene cluster gdmM gene region, after cutting with the BamHI-HindIII enzyme, be cloned into the same loci of pKS (-), obtain recombinant clone pSN101.
PLS406 is cloned into the NdeI-HindIII site of expression vector pET30a (+) from the 1.5kb stuffer that obtains by three fragment connection methods with the cutting of BamHI-HindIII enzyme from the 1.2kb MBP fragment that obtains with from pSN101 with the cutting of NdeI-BamHI enzyme, and obtaining MBP is the fusion expression vector pLS412 of label.
Design primer ALL51:5
'-GGG
GGATCCATGGGGAAAAACTTACAAGCACTG-3', (SEQ ID NO.5), the BamHI restriction enzyme site is represented with underscore; ALL52:5'-GGG
AAGCTT
TTAACTCAAGGCCTCGAATTGTTG-3', (SEQ ID NO.6), the HindIII restriction enzyme site is represented with underscore.(from the Satoshi Tabata professor of Japanese Kazusa DNA institute) is template with anabena Anabaena sp. genomic dna, the ALL51-ALL52PCR amplification obtains 1.2kb N-acetyl-D-glucosamine 2-isomerase gene all3695,1.2kb after cutting with the BamHI-HindIII enzyme, be cloned into the same site of pKS (-), obtain recombinant clone pLS413.PLS413 obtains the fusion expression vector pLS414 of MBP and all3695 with the same loci (namely replacing stuffer) of BamHI-HindIII enzyme cutting from the all3695 gene clone that obtains 1.2kb to pLS412.
Design primer TIG1:5'-GAA
CATATGCACCATCATCATCATCACATGCAAGTT
TCAGTTGAAACC-3', (SEQ ID NO.7), the NdeI restriction enzyme site is represented with underscore; TIG2:5'-GAA
GGATCCCATGGCGCCCTGAAAATAAAGATTCTCCGCCTGCTGGTTCA
TCAGCTC-3', (SEQ ID NO.8), BamHI and NcoI restriction enzyme site are represented with underscore.(from the John Cronan professor of U.S. University of Illinois at Urbana-Champaign) is template with intestinal bacteria MG1655 genomic dna, the TIG1-TIG2PCR amplification obtains 1.3kb tig chaperone gene, 1.3kb be cloned into the precious biotech firm of pMD-18T(, Dalian), obtain recombinant plasmid pLS415.
Design primer MTS1:5'-GAA
GAATTCCCATGGGGATGACTAATCCTCTTGAT
TTTAATATC-3', (SEQ ID NO.9), BamHI and NcoI restriction enzyme site are represented with underscore; MTS2:5'-GAA
GCGGCCGCTCAGTCGACTACCATATCAAATGAAAG-3', (SEQ ID NO.10), the NotI restriction enzyme site is represented with underscore.(from the Sallie Chisholm professor of Massachusetts Institute Technology) is template with proto green algae Prochlorococcus marinus MIT9313 genomic dna, the 1.0kb neuraminic acid synthase gene PMT0106 of MTS1-MTS2 amplification, 1.0kb cut rear clone to the EcoRI-NotI site of pKS (-) with the EcoRI-NotI enzyme, obtain recombinant clone, called after pLS416.PLS415 with the cutting of NdeI-NcoI enzyme from 1.3kb tig gene and pLS416 with the cutting of NcoI-NotI enzyme from 1.0kb PMT0106 gene be cloned into the NdeI-notI site of expression vector pET30a (+) by three fragment connection methods, obtain the fusion expression vector pLS417 of chaperone gene tig and PMT0106.
Design primer HR1:5'-GGG
GGATCCATGGAGAAAGAGCGAGAGACTCTG-3', (SEQ IDNO.11), the BamHI restriction enzyme site is represented with underscore; HR2:5'-GGG
AAGCTTTT
ATTCCGCGCCTCGGCAGGCGGGG-3', (SEQ ID NO.12), the HindIII restriction enzyme site is represented with underscore.Be template with the total mRNA of people, the HR1-HR2 reverse transcription PCR obtains hRnBP gene (the RBP gene of 1.2kb, coding N-acetyl-D-glucosamine 2-isomerase), after 1.2kb cut with the BamHI-HindIII enzyme, the same loci that is cloned into pKS (-) obtained recombinant plasmid pKnBP.PKnBP with the cutting of BamHI HindIII enzyme from the 1.2kb same loci (namely replacing stuffer) that is cloned into pLS412 obtain the fusion expression vector pLS418 of MBP and hRnBP.
The structure of embodiment 2.TEV proteinase gene integrated plasmid
Design primer R6K1:5'-GGG
TCTAGAGCTCTCGAGATATCTATGGACAGCAAG
CGAACCGG-3', (SEQ ID NO.13), the XbaI enzyme cutting site is represented with underscore; R6K2:5'-G
GG
GAATTCGGATCCGGTACCACTAGTTCAGAAGAACTCGTCAAGAAG-3'，
(SEQ ID NO.14), the EcoRI restriction enzyme site is represented with underscore.Be masterplate with pKD4, R6K1-
R6K2PCR amplification 1.1kb kalamycin resistance gene fragment after 1.1kb cuts with the XbaI-EcoRI enzyme, is cloned into the same loci of pKS (-), obtains recombinant clone pLS1913.PLS1913 with the cutting of XbaI-EcoRI enzyme from 1.1kb fragment and 2.1kbpMOD4RT-G with the enzyme cutting from XbaI-EcoRI be connected, transformed into escherichia coli BW25141, screen with the kantlex of 30 μ g/ml and the penbritin of 50 μ g/ml, obtain recombinant clone pR6KMCS.PR6KMCS uses the R6K replicon, and the host bacterium is the intestinal bacteria BW25141 that contains the pir gene, the plasmid that contains R6K replicon reproducible not in e. coli bl21 (DE3), and this has just removed the background interference of plasmid.
According to the TEV proteinase gene sequence of having reported is given birth to the synthetic 0.7kb of worker Bioisystech Co., Ltd in Shanghai TEV gene, be cloned into the BamHI-HindIII site of pKS (-), institute's DCRP is pLS966.PLS966 is with the TEV gene of BamHI-HindIII enzyme cutting from the 0.7kb that obtains, and the same loci (namely replacing stuffer) that is cloned into pLS412 obtains MBP-TEV fusion vector pLS972.PLS972 with the cutting of ClaI-PvuII enzyme from 4.7kb fragment and pR6KMCS with the cutting of ClaI-HincII enzyme from 2.0kb be connected after, transform BW25141, screening obtains pLS1919.
Design primer R1061:5'-GAA
CTCGAGTCTTGAAATAAGATCACTACCG-3', (SEQ IDNO.15), the XhoI restriction enzyme site is represented with underscore; R1062:5'-GAA
CTCGAGTT
ACGCCCCGCCCTGCCACTC-3', (SEQ ID NO.16), the XhoI restriction enzyme site is represented with underscore.Be masterplate with pBAD322C, the chloramphenicol resistance gene that obtains 0.7kb of R1061-R1062PCR amplification, 0.7kb cuts rear clone to the XhoI restriction enzyme site of pLS1919 with the XhoI enzyme, obtains recombinant clone pLS1928, and the host bacterium is BW25141.
Design primer R1067:5'-GGG
GGTACCGCTTGCCAGGAGCGATCTAAC-3', (SEQ ID NO.17), the KpnI restriction enzyme site is represented with underscore; R1068:5'-GGG
TACGTATGCTGTGA
AATGCCGGATGCGG-3', (SEQ ID NO.18), the SnaBI restriction enzyme site is represented with underscore; R1069:5'-GGG
TACGTAGAATTGGCGGTAATGTGGAGATG-3', (SEQ ID NO.19), the SnaBI restriction enzyme site is represented with underscore; R1070:5'-GGG
GTCGACGGGGATAG
AGCGCGTAAGACTG-3', (SEQ ID NO.20), the SaII restriction enzyme site is represented with underscore.Be template with BL21 (DE3) genomic dna, obtain malE upstream region of gene 0.5kb with the R1067-R1068PCR amplification respectively, obtain malE gene downstream 0.5kb with the R1069-R1070PCR amplification.Upstream 0.5kb and downstream 0.5kb are cloned into pMD18-T Simple carrier (the precious biotech firm in Dalian) respectively and obtain recombinant plasmid pLS1937 and pLS1938.PLS1937 with the cutting of KpnI-SnaBI enzyme from 0.5kb and pLS1938 with the cutting of SnaBI-SaII enzyme from 0.5kb be connected by three fragments, be cloned into the KpnI-SaII site of pKS (-), obtain recombinant plasmid pLS2429.
Design primer R1071:5'-CCATACCCACGCCGAAACAAG-3', (SEQ ID NO.21);
R1072：5'-AAGGGGTTATGCTAGTTATTG-3'，（SEQ?ID?NO.22）。Be template with pLS1928, the R1071-R1072PCR amplification obtains the 3.0kb fragment, and blunt end is cloned into the SnaBI site of pLS2429 then, obtains recombinant plasmid pLS2430.PLS2430 with the cutting of KpnI-SaII enzyme from 4.0kb fragment and pR6KMCS with the cutting of KpnI-SaII enzyme from 2.0kb be connected, transforming the host bacterium is BW25141, screening obtains final recombinant clone pLS2431.PLS2431 is both sides and contains 500bp at containing of malE zone of the plasmid T7-malE-TEv-cat box gene, the R6K replicon.
The inventive method synoptic diagram adds shown in the accompanying drawing 1.The malE-TEV fusion gene and the chloramphenicol resistance gene cat that driven by T7 that contain homology arm are coming under the recombinase catalysis of lambda particles phage, be integrated into the genome of e. coli bl21 (DE3) by homologous recombination, malE Gene Partial on the genome is knocked out, and obtains genetic engineering recombination strain LS2401.LS2401 expresses the MBP-TEV fusion rotein under IPTG induces, thereupon, fusion rotein takes place from shearing, and TEV proteolytic enzyme discharges, and passes through affinity chromatography and obtain purifying.The note of title is as follows among the figure: H1 and H2 are homologous fragment; Neo is kalamycin resistance gene; MalE is the maltose binding protein gene; Cat is chloramphenicol resistance gene; T7 is the T7 promotor, and S is the TEV restriction enzyme site, and Ni-NTA represents affinity chromatography.
1. isopropyl-(IPTG) induces preparation and the electricity of the bacterial strain electricity transformed competence colibacillus cell of recombinase expression to transform.
At first according to a conventional method plasmid pTKRed is converted into e. coli bl21 (DE3), with 100 μ g/ml
Spectinomycin screens under 30 ° of C.Single bacterium colony of obtained strains is forwarded in the LB liquid culture that contains 100 μ g/ml spectinomycins, 30 ° of C overnight incubation, 1ml is forwarded to the same substratum of 100ml, 30oC, shaking culture about 0.2 o'clock to OD600, add 2mM IPTG and induce, to OD600 about 0.4 o'clock, bacterium liquid is poured into the centrifuge tube of precooling, ice bath 10 minutes, 4 ° of C, centrifugal 5 minutes of 7000rpm abandons supernatant.Wash thalline 3 times with 10% ice-cold glycerine, suspend with 10% ice-cold glycerine of 200 μ l at last, 50 μ l packing also are used for once electric the conversion.
PLS2431 with the cutting of KpnI-SaII enzyme from 4.0kb T7-malE-TEV-cat fragment, be dissolved in ddH
2O, 1 μ g DNA add to the competent cell that electricity that above-mentioned recombinase expresses transforms 50 μ l, flick mixing.Mixed solution is transferred in the 1mm electricity revolving cup of precooling on ice, electric shock transforms.Electricity conversion condition: 1.8kV, 200 Ω, the Gene Pulser II of U.S. Bio-Rad company
RThe electricity conversion instrument.After electricity transforms, after the suspension of 1mL SOC substratum, go to the 15ml polypropylene centrifuge tube, the 37oC shaking culture is cultivated 2h, is applied to the LB flat board of the paraxin that contains 25 μ g/ml, and screening obtains recombinant bacterial strain.
2. the evaluation of integrated engineering strain
Design primer R1073:5'-GAGCGCCAGTTGCCACTCATC-3', (SEQ ID NO.23) is the primer of integration site upstream 58bp; R1074:5'-ACGCGTTGGTTAATCACCTC-3', (SEQ ID NO.24) is the primer of integration site downstream 76bp; R1075:5'-AGTCTGCGCGTCTTTC
AGGGCTTC-3', (SEQ ID NO.25) is the primer at malE gene 3' end.Carry out bacterium colony PCR with the chlorampenicol resistant bacterial strain and verify its genotype, checking the results are shown in accompanying drawing 2.Be template with former strain BL21 (DE3) genome, the R1073-R1074PCR amplification be the 2.4kb fragment; Be template with the chlorampenicol resistant bacterial strain, the R1073-R1075 amplification obtains the upstream integration site to the 1.9kb fragment of inserting between the fragment, the R1061-R1074 amplification obtains inserting fragment to the 1.5kb fragment between the site, downstream, and the R1073-R1074 amplification obtains the full length fragment of 4.1kb.
T7-malE-TEV-cat replaces the bacterial strain called after LS2401 malE gene region, the correct gene type on e. coli bl21 (DE3) genome, LS2401 is kept at (No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center as patented strain on April 17th, 2013, Institute of Microorganism, Academia Sinica), classification called after colon bacillus (Escherichia coli), deposit number is CGMCC No.7482.
Expression and the purifying of embodiment 4. integrated TEV proteinase genes
The LS2401 bacterium colony is forwarded in the LB liquid culture, and after 37 ° of C overnight incubation, 1ml is forwarded to 50ml LB substratum, 37 ° of C, and shaking culture about 0.7 o'clock to OD600 adds 1mM IPTG and continues to cultivate shaking culture 6h under 30 ° of C, centrifugal collection thalline.The step of carrying out affinitive layer purification TEV with the U.S.'s Ni-NTA of Novagen company resin is as follows.Add 1.0ml lysis buffer (50mM NaH
2PO
4, 300mM NaCl, the 10mM imidazoles, pH8.0) suspension bacterial sediment, in ultrasonication on ice (10 seconds * 6 times, 5 seconds at interval) with lysing cell.4 ° of C thereupon, 10,000g, 30min, centrifugal, the Ni-NTA mixed with resin of getting cleer and peaceful 150 μ l, 4 ° of C 3h that vibrates gently, centrifugal, abandon supernatant.Add 600 μ l washings (50mM NaH
2PO
4, 300mM NaCl, the 20mM imidazoles, pH8.0) washing resin is three times, divides at last three times, and each is with 200 μ l elution buffer (50mM NaH
2PO
4, 300mM NaCl, the 250mM imidazoles, pH8.0) and the Ni-NTA mixed with resin, and 4 ° of C 3h that vibrates gently, centrifugal, get supernatant, and merge albumen.SDS-PAGE detects, and the results are shown in accompanying drawing 3.The total protein that LS2401 expresses, total length 69.6KD, owing to obtain the MBP of 41.0KD and the His-TEV of 28.6KD from shearing in the cell, as seen about 50% albumen is that form with soluble proteins exists.One step Ni-NTA resin affinity chromatography can be purified to TEV the purity more than 95%, and with the protein content of Bradford method mensuration purifying, the output that calculates TEV is 4.2mg/L.Embodiment 5. Expression of Fusion Protein, purifying and TEV enzyme are cut
Respectively the fusion expression vector pLS414 of MBP and all3695 and the fusion expression vector pLS417 of chaperone gene tig and PMT0106 are expressed by above-mentioned method, and press Ni-NTA method purified fusion protein.Fusion rotein carries out enzyme with the TEV proteolytic enzyme of above-mentioned purifying to be cut, and enzyme is cut system: 20 μ g albumen, and 0.5 μ g TEV, damping fluid is 50mM Tris-HCl, pH8.0,0.5mM EDTA, 1mM DTT.Cumulative volume 150 μ l, 30 ° of C enzymes are cut 3h.
What TEV proteolytic enzyme enzyme was cut the His-MBP-hRnBP fusion rotein the results are shown in accompanying drawing 4.The His-MBP-hRnBP fusion rotein of expressing is 89.8KD, after the TEV enzyme is cut, obtains the hRnBP albumen of 48.1KD and the His-MBP of 41.7KD.The SDS-PAGE that TEV proteolytic enzyme enzyme is cut the His-TIG-PMT0106 fusion rotein the results are shown in accompanying drawing 5.The His-TIG-PMT0106 fusion rotein is 88.9KD; After the TEV enzyme is cut, obtain the His-TIG of 49.9KD and the PMT0106 of 39.0KD.
As seen from the figure, TEV proteolytic enzyme can act on the restriction enzyme site between fusion tag and the target protein and the two is separated effectively, fails enzyme to cut entirely, may be that the enzyme tangent condition is not best reason.The target protein that separates can further separate by column chromatography and the label protein that contains Histag and TEV albumen.Therefore, can be used for the highly active TEV proteolytic enzyme of separation and purification tool efficiently based on genomic TEV proteolytic enzyme expression system, the shearing certainly of MBP-TEV in express cell also proved the high reactivity of TEV proteolytic enzyme.
Claims (3)
1. a TEV proteolytic enzyme is expressed engineering bacteria, is colon bacillus (Escherichia coli) CGMCC No.7482.
2. the described TEV proteolytic enzyme of claim 1 is expressed the construction process of engineering bacteria, it is characterized in that be will be subjected to the homologous recombination method that mediates by the recombinase that derives from lambda particles phage of the TEV proteinase gene that merges with maltose binding protein gene malE that drives of T7 strong promoter and chloramphenicol resistance gene be integrated in escherichia coli expression bacterial strain BL21 (DE3) genome, and replace the malE gene region in the genome and obtain.
3. the described TEV proteolytic enzyme of claim 1 is expressed the application of engineering bacteria in expressing TEV proteolytic enzyme.
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