The Construction And Expression Of The Prokaryotic Expression Vector About The Multi-copy Recombinant Tandem Metallothionein Gene From Eriocheir Sinensis

For Metallothionein(MT) contains many Cys residues(above 30%) and that those Cys residues keep reducing with bonding metal,theMT have some special biological functions ,e.g : antioxidation,geting rid of free radical and heavy metal detoxication.Thus aroused in scientific research high level of enthusiasm, in practical application has a very high value, especially in medicine, health care and other fields. But nowadays the metallothionein is mainly from animal organs extracts, with some difficulties i.g: microamount extraction,high preparation costs , some degree of heavy metal pollution. To solve this problem, we constructed a set of prokaryotic expression systems by pET-15b vector and MT cds which including 1 copy, 2 copies and 3 copies of the Eriocheir sinensis metallothionein gene.After exploring the best cultivation and induction conditions, we successfully and efficiently obtained MT with 1copy, 2 copies and 3 copies. The target protein expressed mainly in the form of inclusion bodies and account for the total bacterial protein about 40%, which resolved the difficulty above and made a step for the research of the physical and chemical properties, biological function and application of MT. We also obtained the MT dfficiently by the fermentation technology in fermentor (Biostat Bplus 5.0L).This verified the large-scale industrial preparation of the feasibility of the Metallothionein. Meanwhile, in order to obtain single target protein, we added specific cleavage sites between the copies of MT when we design the primers. But the cutting results show that it is not so easy to do that. We suppose that maybe the MT constructed intramolecular or intermolecular disulfide bridge structure, which interfered the cutting result. We dissolved the inclusion bodies with 6M guanidine hydrochloride and used guanidine hydrochloride concentration gradient dialysis (4M, 2M guanidine hydrochloride, 0.01M PBS) to refold. Then check the refolded protein with DPPH . The results show that the refolding condition should be further improved.ObjectiveTo construct multi-copy tandem repeat prokaryotic expression vector of the Crab Metallothionein gene and express it efficiently。To refold the inclusion body if necessaryMethod1. Construction of the Metallothionein gene prokaryotic expression vectors pET-2MT,pET-3MT and the genetic engineering bacteria which contains 2 and 3 copies of the MT gene.(1) Design primers: Due to insert multiple copies of the target gene cds, when primers were designed we considered the insertion order for those fragments for they were inserted one by one. In the design of the first pair of primers, in addition to add a restriction enzyme site in the upstream primer, there were two restriction enzyme sites in the downstream primer which specially provided conditions for the second target fragment to be serted into vector.similarly, in the second pair of the primer there were also two restriction enzyme sites in the downstream primer for insertion of the third segment. A specific protein cleavage site was added in each downstream primer 5 'to meet the requirements if necessary that can cut the multi copies target protein into monomers.(2) Conventional PCR: Did conventional PCR with 3 pairs of primers respectively and the recombinant plasmid GST-MT (constructed by the Research Center of Experimental Medicine of Guangzhou Medical University).Obtained 3 fragments of MT gene CDS The differences among the 3 fragments were the restriction enzyme sites. According to the insertion order in the requirements, the fragments was marked for the f1,f2,f3.(3) T-A cloning: Respectively using the three fragments obtained above with pMD19-T Simple Vector did T-A cloning and transformed into E. coli BL-21. Select positive clones, did restriction enzyme digestion and DNA sequencing. If the result was correct, marke it as T1, T2, T3 corresponding to the f1,f2, f3.(4) Subcloning: Using the prokaryotic expression vector pET-15b to establish metallothionein gene expression vector of tandem repeats with 6 His tag . To cut vector T1 and pET-15b with selected restriction enzymes Group 1, gel purified the target fragments and ligate them with T4 DNA ligase.Then transformed into E.coli BL21(DE3)pLysS. Select positive clones, did restriction enzyme digestion and DNA sequencing. If the result was what we want, marked as pET-1MT. Similarly cut vector T2 and pET-1MT with selected restriction enzymes Group 2, gel purified the target fragments and ligate them with T4 DNA ligase. Then transformed into E.coli BL21(DE3)pLysS. Select positive clones, did restriction enzyme digestion and DNA sequencing. If the result was correct, marked as pET-2MT.Similarly construnctored the pET-3MT,which contains 3 copies of the MT gene cds.2. Prokaryotic expressionAfter the Exploration of the best expression conditions ,put the constructed genetical engineered bacteria in the best conditions to express the MT.The best conditions are : LB medium [1% (m / v) Trytone, 0.5% (m / v) Yeast Extact, 1% (m / v) NaCl, 60μg/ml AMP, 300uM ZnCl2], keep 37℃, 300r/min temperature oscillation shaking culture to cell OD value between 0.4-0.5, added 1.0mM IPTG to a final concentration to induce the protein expression for 8h. 3. SDS-PAGE analysis of expression products Collected the fermentation supernatant, the cell supernatant and the precipitation after ultrasonic disruption.Did 12% separating gel SDS-PAGE, observed the target band.4. Western blot analysis of expression products Collect the fermentation supernatant, the cell supernatant and the precipitation after ultrasonic disruption, with rabbit anti-His (His-probe) did western blot.5. Inclusion body purification: collected fermentation of the bacteria by centrifugation in low-temperature.Collected the precipitation after ultrasonic disruption in ice.Then collect inclusion bodies by low-temperature high-speed centrifugation. Wash the inclution bodies by 4M urea and centrifuge it in high-speed.6. Large-scale preparation of the recombinant MT by fermentor.7. Refolding inclusion bodies. inclusion bodies was dissolved in 6M guanidine hydrochloride, adding DTT to a final concentration of 200mM, in4℃environment through 4M, 2M guanidine hydrochloride concentration gradient dialysis, and then by 0.1M PBS dialysis8. DPPH colorimetric method check the protein refolding activity: DPPH colorimetric method was used to check the antioxidant activity of metallothionein, to detect the effect of protein refoldingResult1,We Successfully constructed a set of copies of metallothionein gene expression vector and transformed into E. coli BL21 (DE3) pLysS.Those tandem repeat contais 1 copy,2 copies,3 copies MT gene cds.The prokaryotic expression system expressed the target protein successfully and efficiently. 2,high purity inclusion body could obtained by 4M urea washing with3,SDS-PAGE electrophoresis show that the target proteins were mainly from the precipitation after ultrasonic disruption and in the form of insolution body.4,Western blot analysis: There were target proteins in the precipitation after ultrasonic disruption. MT proteins form polymers easily.5,We obtained the MT dfficiently by the fermentation technology in fermentor (Biostat Bplus 5.0L).6,PDDH determination the refolding show that the refold conditions should be further improved.Conclusion1. We successfully constructed multi-copy crab metallothionein tandem repeat gene cds prokaryotic expression system and efficiently expressed the target protein, which lay a solid foundation for further study about the MT.2. We obtained the MT dfficiently by the fermentation technology in fermentor (Biostat Bplus 5.0L). This verified the large-scale industrial preparation of the feasibility of the Metallothionein.2. We found that the multi-copy metallothioneinextracted from prokaryotic expression system , unlike many kinds of protein,could form complex molecules intramolecular or intermolecular.This suggest that the physical,chemical and abiochemical properties of metallothionein are more complex than what we have got. It is necessary to do more research about MT. And the inclusion body refolding conditions need further improvement..