CN1844398A - Fusion expression method for metallothionein and use thereof - Google Patents
Fusion expression method for metallothionein and use thereof Download PDFInfo
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- CN1844398A CN1844398A CN200610040179.9A CN200610040179A CN1844398A CN 1844398 A CN1844398 A CN 1844398A CN 200610040179 A CN200610040179 A CN 200610040179A CN 1844398 A CN1844398 A CN 1844398A
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Abstract
The invention attribute to bioengineering technical field, the described new method that use the metallothionin as fusion label to fuse and express together with exogenous gene has the characteristic in expressing target product yield solubility, high productive and stability, separating and purifying the target protein conveniently through metal chelating affinity chromatography. The fusion expression system applies to the study of bioengineering pharmaceutics, gene engineering, chemical biology, molecular biology. Fusion expression vector pT7MT is a highly effective fusion expression vector that can express the metallothionin solubility and high productive in bacillus coli.
Description
One, technical field
The invention belongs to technical field of bioengineering.
Two, background technology
Utilize genetic engineering technique to produce the method that recombinant protein has become widespread use in biotech medicine product industry and genetically engineered, biological chemistry and the molecular biology research.For the ease of efficiently expressing and the separation and purification in downstream of recombinant protein, at present, a lot of fusion expression vectors have been developed in order at the expression in escherichia coli fusion rotein.Studies show that, compare that the strategy of amalgamation and expression can increase output, solubility and the homogeneity of target protein with non-fusion expression.And, merge and often can also improve the stability of target protein in expression system at N-terminal fusion tag.In addition, because the affinity of fusion tag, method that can be by affinity chromatography purifying easily obtains highly purified Target Fusion albumen.In addition, with foreign gene and can be in intestinal bacteria high level expression and the good protein of product solvability (as staphylococcus aureus protein A, glutathione-S-transferase, intestinal bacteria maltose binding protein malE, escherichia coli thioredoxin etc.) be built into the form of fusion rotein, good by means of solubility property, the fusion partner protein of high level expression makes needed target protein (being exogenous genes products) also obtain the great expression of solubility, thereby avoids the generation of inclusion body products.More than these methods all found and set up, and all obtained patent of invention by external scientist.Wherein, current most popular two kinds of affinity tag are glutathione-S-transferase (GST) and His6-label, a lot of commercialization carriers are developed the target protein that is used for expressing the fusion form with GST or His6-label, and then use gsh affinity chromatography or Ni post affinity chromatography to obtain purifying.
(metallothione MT) is the intracellular protein that amino acid a kind of small molecular weight, 30% is cysteine residues to metallothionein(MT), and its physiological action may be to play oxidation resistant effect by removing hydroxide ion and bind metal ion.Metallothionein(MT) is rich in cysteine residues, can be in conjunction with a plurality of zine ions and cupric ion under physiological condition.In single celled eukaryote, metallothionein(MT) is preferentially in conjunction with cupric ion; Then preferentially in conjunction with zine ion, and zine ion also may be replaced by cupric ion or calcium ion in mammalian cell.
Three, summary of the invention
The objective of the invention is to invent a kind of fusion expression vector based on the metallothionein(MT) fusion tag, abundant and enhancing human means and ability by genetic engineering technique expression, purification of Recombinant target product, similar approach has never seen report at home and abroad.
Purpose of the present invention can reach by following measure:
Metallothionein gene is placed under the control of promotor commonly used, after the metallothionein gene encoding sequence, insert multiple clone site.In multiple clone site, design has common protease site between metallothionein(MT) and target protein matter, so that target product is discharged from fusion rotein with the target protein gene clone.After metallothionein(MT)-target product fusion rotein is expressed in gene engineering expression system commonly used, utilize the sequestering action of metallothionein(MT) and divalent-metal ion, can take the method for metal chelate chromatography that fusion rotein is carried out purifying, utilize common proteolytic enzyme that target product is discharged then and separate.Metallothionein(MT) also can be used as the fusion of fusion rotein label and expresses at the carboxyl terminal (and downstream of target product) of target product, fusion expressed product can utilize the metal chelate chromatography method to carry out purifying, utilizes common proteolytic enzyme that target product is discharged then and separates.
Characteristic of the present invention is metallothionein(MT) is incorporated in the gene engineering expression carrier commonly used as fusion tag, and its result can be so that target protein have obtained stable, soluble efficiently expressing; And because metallothionein(MT) itself has the ability of bind metal ion, therefore can be used as a kind of affinity tag is used for expressing and purification of target albumen, and increased the stability and the solubility of target protein, improved the productive rate that bioactive expression product is arranged.
Purposes of the present invention is the gene engineering expression system that metallothionein(MT) can be applied to use always as general, effective, an easy fusion tag, improve the output that bioactive recombinant protein is arranged, make things convenient for the separation and purification of target product, be applicable to researchs such as biotech medicine product industry and genetically engineered, biological chemistry, molecular biology.
Four, description of drawings
Fig. 1. recombinant expression plasmid pT7MT synoptic diagram.
1:colE1 plasmid replication starting point; 2:Lac I gene; The 3:T7 promotor; 4: the Ω sequence; 5: ribosome bind site and SD sequence; 6: metallothionein(MT) 2A gene; 7: multiple clone site; The 8:f1 replication origin; 9: ampicillin resistance gene.
Fig. 2. the SDS-PAGE of reorganization MT-GST expressing fusion protein and purifying analyzes.
(A) MT-GST Recombinant Protein Expression and purifying
1. molecular weight standard protein: be respectively 97,66,43 from top to bottom, 31kDa;
2. through IPTG inductive pT7MT-GST/BL21 (DE3) lysis supernatant;
3.Ni
2+Affinitive layer purification pass the peak;
4.250mM the elution peak of imidazoles.
(B) GST-MT Recombinant Protein Expression and purifying
1. molecular weight standard protein: be respectively 97,66,43,31 from top to bottom, 17kDa;
2. through IPTG inductive pTORG-MT/BL21 (DE3) lysis supernatant;
3. the elution peak of gsh affinity chromatography 10mM GSH;
4.Sephadex G-15 removes the collection peak behind the imidazoles.
(C) comparison of reorganization MT-GST albumen and reorganization GST-MT protein expression
1. molecular weight standard protein: be respectively 97,66,43,31 from top to bottom, 17kDa;
2. through the GST-MT of IPTG abduction delivering lysis supernatant;
3. through the MT-GST of IPTG abduction delivering lysis supernatant.
Comparison that Fig. 3 .pET28a-Tn I and pT7MT-Tn I express and the purifying of MT-Tn I
(A) Tn I-His6 Recombinant Protein Expression
1. molecular weight standard protein: be respectively 97,66,43,31 from top to bottom, 17kDa;
2. through the gross protein of IPTG inductive pET32a-Tn I/BL21 (DE3) cell;
3. through the ultrasonic degradation supernatant of IPTG inductive pET32a-Tn I/BL21 (DE3) cell;
4. the inclusion body behind the ultrasonic degradation of IPTG inductive pET32a-Tn I/BL21 (DE3) cell.
(B) MT-Tn I Recombinant Protein Expression
1. molecular weight standard protein: be respectively 97,66,43 from top to bottom, 31kDa;
2. through the gross protein of IPTG inductive pT7MT-Tn I/BL21 (DE3) cell;
3. through the ultrasonic degradation supernatant of IPTG inductive pT7MT-Tn I/BL21 (DE3) cell;
4. the inclusion body behind the ultrasonic degradation of IPTG inductive pT7MT-Tn I/BL21 (DE3) cell.
(C) purifying of MT-Tn I recombinant protein
1. molecular weight standard protein: be respectively 97,66,43 from top to bottom, 31kDa;
2. the lysis supernatant after the reorganization MT-Tn I protein induced expression;
3.Ni
2+The reorganization MT-Tn I that affinitive layer purification obtains.
Five, embodiment
1.pT7MT structure:
PT7MT is at pTORG (Chinese invention patent: ZL01127171.X) make up on the basis of prokaryotic expression carrier.By NcoI and BamH1 double digestion, the GST encoding sequence in original pTORG carrier is removed, and then is replaced by the encoding sequence of MT2A.By PCR method, originally the thrombin restriction enzyme site in the carrier is replaced by factor Xa.
The pcr template of MT2A is that human heart library (Clontech company) is template, the PCR primer is as follows: upstream primer: 5 '-CAT GCC ATG GAC TGC TCC TGC GCC GCC-3 ', downstream primer: 5 '-CGGGAT CCC ACG ACC TTC GAT GGC GCA GCA G-3 '.The MT2A gene fragment that amplification obtains reclaims through agarose gel electrophoresis, carry out Nco I and BamH I double digestion then, again with is connected through the pTORG after same the processing after, through transforming and screening acquisition expression vector pT7MT, as shown in Figure 1, its sequence is: T7 promotor-LacO-TCTAGATATTTT
TACAACAATTACCAACAACACAAACAACAAACAACATTACA
The Ω leader sequence
Initiator codon
ATTACTATTTACAATAACAATGGCTAGAAATAATTTTGTTTAACTTTAAG
AAGGAGATATACC
ATGGAA
Ribosome bind site and SD sequence A CAGTATTC-MT2A encoding gene-
GGGATCCCCGGGAATTCGAGCTCCGTCGACAAGC
Factor Xa recognition site encoding sequence multiple clone site
TTGCGGCCGCACTCGAG 
---T7 terminator
Terminator codon
2. the structure of fusion expression vector pT7MT-GST and pT7MT-Tn I:
Obtain the encoding sequence of GST and human troponin (Troponin I, Tn I) by pcr amplification, be inserted into and obtain corresponding fusion expression vector pT7MT-GST and pT7MT-Tn I among the pT7MT.The PCR primer of gst gene is as follows: upstream primer: 5 '-CGCGGATCCATGGCCAGCTACCGAC-3 ', downstream primer: 5 '-TCCCTCGAGTTATACGACAAAGCGGG-3 '; The PCR primer of Tn I is as follows: upstream primer: 5 '-CGCGGATCCAAATGGGAGATGAGGA-3 ', downstream primer: 5 '-GCCCTCGAGCTATTGTGAGGTCGGAGA-3 '.Be inserted among the pT7MT behind PCR fragment process BamH I that obtains and the Xho I double digestion, obtain MT-GST and MT-Tn I fusion expression vector pT7MT-GST and pT7MT-Tn I through screening.Simultaneously, we have made up the GST-MT/pTORG expression vector.With human heart library (Clontech) is that template amplification obtains the MT2A gene, and primer is as follows: upstream primer: 5 '-CCGGGATCC TCA TAT GGC CAT GGA-3 ', downstream primer: 5 '-CGG CTC GAG TCA CAT TAT TTC ATA GA-3 '.The MT2A gene is handled the back, is inserted into and obtains pTORG-MT among the pTORG through BamH I and Xho I.Another one non-fusion expression carrier pET28a-Tn I makes up and preservation [Southeast China University's journal (medicine), 24 (1): 36-38,2005] by this laboratory is previous.
3.MT-GST, MT-Tn I and GST-MT Recombinant Protein Expression:
Recombinant Protein Expression: the picking mono-clonal is in the LB substratum, and 37 ℃, the 250rpm jolting is spent the night.Be transferred in the fresh LB substratum that is added with 100 μ g/ml penbritins by 1: 100 in second day, 37 ℃, 250rpm jolting 3-4 hour, up to OD600 about about 1.0, adding final concentration is the IPTG abduction delivering of 0.1mM, total induction time 4 hours, centrifugal 5 minutes results of 6000rpm thalline.
4.MT-GST, the purifying of MT-Tn I and GST-MT recombinant protein:
For MT-GST and two protein of MT-Tn I, will be suspended in 50ml 50mM PBS from total thalline of 1 liter of substratum again, pH8.0, ultrasonic 30 minutes smudge cellses.Centrifugal 20 minutes of 12000g separates ultrasonic supernatant.Purifying is according to His-Select TM HC Ni
2+The operation instruction of affinity media (Sigma company) is carried out.At first with Ni
2+Affinity media carries out balance with level pad (50mM PBS, pH8.0,10mM imidazoles), then with sample on the ultrasonic supernatant.Use level pad (50mM PBS, pH8.0,10mM imidazoles) to be washed till baseline behind the last sample again, use elution buffer (50mM PBS, pH8.0,250mM imidazoles) to carry out wash-out at last.Imidazoles in the elution peak component is removed by Sephadex G-15 gel-filtration.The albumen that final purification obtains is frozen in-70 ℃.
Using method according to the GST affinity chromatography medium is carried out purifying, obtains the GST-MT albumen of purifying.Total thalline from 1 liter of substratum is suspended in 50ml PBS (137mM NaCl, 2.7mM KCl, 10mMNa again
2HPO
4And 2mM KH
2P0
4PH 7.4), ultrasonic 30 minutes smudge cellses.Centrifugal 20 minutes of 12000g separates ultrasonic supernatant.Then with affinity column on the ultrasonic supernatant.Be washed till baseline with level pad PBS again behind the last sample, use elution buffer 10mM gsh (using 50mM Tris-HCl, pH 8.0 preparations) to carry out wash-out at last.
In the purge process protein concentration of all components to adopt BSA be standard substance as with reference to, carry out quantitatively with the BCA test kit, the 12%SDS-PAGE with coomassie brilliant blue staining analyzes simultaneously.
We have obtained the MT2A gene by PCR, and it is cloned in the pTORG carrier, replace original GST and obtain a new expression vector pT7MT (Fig. 1).Use this carrier, we have made up pT7MT-GST and pT7MT-Tn I expression vector, and have expressed MT-GST and MT-Tn I albumen.The e. coli bl21 (DE3) that has transformed the pT7MT-GST plasmid a protein induced band of tangible MT-GST (Fig. 2) occurs in the position of molecular weight 34kDa after IPTG induces.Through Ni
2+After the post affinity chromatography, obtained stable, purity and reached 87% MT-GST fusion rotein, output reaches the 30mg/L nutrient solution.For the ease of relatively, we also express simultaneously and purifying GST-MT albumen.Serious degraded has taken place in the GST-MT behind the abduction delivering in the process of carrying out purifying with GST post affinity chromatography, almost completely GST only is left in degraded at last, so output has only the 1mg/L nutrient solution.By these two proteic comparison shows that, be placed on the C end and express very unsettled MT albumen, place the N end to improve greatly with rear stability, proteic degraded (Fig. 2 A) does not only appear, and may be owing to the stability of the internal structure of MT albumen own, its expression level and solubility have all improved (Fig. 2 C) greatly.
Effective equally for other proteic expression in order to show pT7MT, we have attempted the amalgamation and expression of Tn I.According to former report and our previous studies show that, Tn I albumen is assembled and the formation inclusion body when expression in escherichia coli easily.Studies show that when the proteic carboxyl terminal of Tn I added that a His6-label is expressed, proteic solubility only accounted for 10% (Fig. 3 A) of whole expression amounts before us; The proteic preparation of a large amount of activated Tn I is (the Chinese invention patent application number: 200410014859.4) that becomes the method acquisition of renaturation by external inclusion body at last.And when we after the aminoterminal of Tn I has added the fusion tag of MT2A, the solubility of fusion rotein has reached 60% (Fig. 3 B), passes through Ni
2+Output behind the post affinity chromatography single step purification reaches 28mg/L nutrient solution (Fig. 3 C).This shows that MT2A can be used as a kind of alternate amalgamation and expression companion and improves solubility and the output that target protein is expressed, and can also make things convenient for its purifying simultaneously.
Can know from above-mentioned example and find out that we are that mate molecule and affinity tag have made up a new fusion expression vector pT7MT with MT2A albumen, foreign gene can obtain stably express soluble, high yield when expressing in this expression vector.MT2A is because of itself intrinsic metal ion binding ability, and the fusion tag that can be used as a kind of metal chelate affinity chromatography helps the purifying of target protein.
Claims (8)
1. the method for a genetically engineered metallothionein(MT) amalgamation and expression is characterized in that utilizing metallothionein(MT) to carry out amalgamation and expression together as fusion tag and foreign gene.
2. the method for a kind of genetically engineered metallothionein(MT) amalgamation and expression according to claim 1, it is characterized in that can solubility ground, high place of production stably express target product.
3. the method for a kind of genetically engineered metallothionein(MT) amalgamation and expression according to claim 1 is characterized in that carrying out the separation and purification of target protein by metal chelate affinity chromatography easily.
4. the method for a kind of genetically engineered metallothionein(MT) amalgamation and expression according to claim 1 prepares, produces the application in the recombinant protein in genetically engineered or biotechnology.
5. a bacillus coli gene engineering metal sulfoprotein fusion expression vector pT7MT is characterized in that utilizing metallothionein(MT) effect fusion tag and foreign gene to carry out amalgamation and expression together.
6. a kind of intestinal bacteria metallothionein gene engineering fusion expression vector pT7MT according to claim 5, it is characterized in that can be in intestinal bacteria solubility ground, high place of production stably express target product.
7. a kind of bacillus coli gene engineering metal sulfoprotein fusion expression vector pT7MT according to claim 5 is characterized in that carrying out the separation and purification of target protein by metal chelate affinity chromatography easily.
8. a bacillus coli gene engineering metal sulfoprotein fusion expression vector pT7MT prepares, produces the application in the recombinant protein in genetically engineered or biotechnology.
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| CN102040653A (en) * | 2010-09-07 | 2011-05-04 | 湘潭大学 | Tag peptide capable of realizing affinity binding with polystyrene and method for preparing enzyme-linked immuno solid phase antigen with same |
| CN101875699B (en) * | 2009-11-23 | 2012-06-13 | 上海司睿宝生物科技有限公司 | Fusion protein of human epidermal growth factor and metallothionein and preparation method and application thereof |
| CN103194478A (en) * | 2013-04-16 | 2013-07-10 | 南京大学 | Soluble recombinant insulin, expression method of insulin derivative and application of insulin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6391590B1 (en) * | 1991-10-21 | 2002-05-21 | The Regents Of The University Of California | Recombinant streptavidin-metallothionein chimeric protein having biological recognition specificity |
| GB9506051D0 (en) * | 1995-03-24 | 1995-05-10 | Univ Singapore | Gene expression |
| US5824512A (en) * | 1996-11-22 | 1998-10-20 | The United States Of America As Represented By The Secretary Of The Navy | Bacteria expressing metallothionein gene into the periplasmic space, and method of using such bacteria in environment cleanup |
| AUPR805101A0 (en) * | 2001-10-03 | 2001-10-25 | University Of New South Wales, The | A method of genetic screening using an amplifiable gene |
| CN1644699A (en) * | 2004-12-13 | 2005-07-27 | 东北林业大学 | Preparation for plant metal surfur protein |
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