Research On Expression Of Human TGF-?1 In Escherichia Coli And Pichia Pastoris

Objective:TGF-?1 is one of the members of the TGF-? superfamily,which plays a critical role in the development of tumors and tissue wound healing.In the recent twenty years,scientists have never diminish their passion to TGF-?1.Skin hyperpigmentation is one of the most common skin aesthetic that affects women.Recently,it has been reported that TGF-?1 plays a pivotal role in inhibiting melanin synthesis in adipose-derived stem cells,and another study indicated that TGF-?1 is a potent inhibitor of melanin synthesis,though decreasing the expression level of tyrosinase.Based on the above,inhibition of hyperpigmentation by dermal topical application would be a wide area with potential application of TGF-?1 beyond wound repair.Adopting the means of recombinant techniques to establish efficient preparation method is the foundation of TGF-?1 application.In this experiment,two different recombinant plasmids were contrasted and then transformed into E.coli and Pichia,and used mouse melanoma cells to estabilish a recombinant hTGF-?1 inhibitory melanin production antivity evaluation system.The aim is to establish a complete and efficient method for the preparation of recombinant hTGF-?1,lying the foundation for its development and application in more fields.Method: 1?Construction and expression of recombinant hTGF-?1 in Escherichia coli expression systemE.coli preferred codons to optimize the coding sequence of hTGF-?1,with restriction sites and protective bases introduced at both ends.The target genes was amplified by PCR,and along with expression vector pET21 b were digested simultaneously by double enzyme digestion method,and the target gene was inserted into a corresponding site of the expression vector to obtain a recombinant.The recombinant plasmid was transformed into E.coli DH5? and further transformed into expression host E.coli BL21(DE3).The recombinant engineering strains with high expression were screened by reducing SDS-PAGE.After mass fermentation,the inclusion bodies were broken by ultrasonication and purified by weak cation exchange chromatography and size exclusion chromatography,and then the inclusion bodies were renatured by dilution.2?Construction and expression of recombinant hTGF-?1/C77 S in Pichia yeast expression SystemOnline optimizing hTGF-?1/C77 S coding sequence using Pichia yest preference codons,introducing protective bases,restriction sites,and signal peptide sequence genes at the beginning,and inserting His tags,restriction sites,and protecting bases at the end.The target gene was amplified by PCR and the recombinant plasmid pGAPz?A-hTGF-?1/C77 S was constructed by double enzyme digestion.The linearized recombinant plasmids were digested and transformed into the expression host Pichia X33 by electroporation.High copy recombinants were screened by contains high concentrations of antibiotics plates and identified by PCR and analyzed by SDS-PAGE,protein dot blot and Weston Blot.3?Biological activity detection of recombinant hTGF-?1The effect of hTGF-?1 prepared on the cell viability of B16-F10 mouse melanoma cell was detected by MTT assay,and the bioactivity of melanin production in B16-F10 cells was detected by alkaline lysis assay.Results: 1?Using the E.coli expression system,hTGF-?1 coding sequence was successfully cloned into expression vector and screened for highly expressed engineering strains.Purification of inclusion bodies was carried out using two chromatographic steps reaching 95% purity,and a stable hTGF-?1 solution was obtained after dilution and renaturation.2?Using the Pichia yeast expression system,the hTGF-?1/C77 S coding sequence was successfully cloned into the expression vector pGAPz?A and transformed into Pichia yeast X33.The results of Weston Blot showed that the recombinant monomer hTGF-?1 could be effectively expressed in Pichia pastoris.About 20% of the total expression of target protein could be effectively secreted to the extracellular.3?Recombinant hTGF-?1 was successfully verified that it has melanin production inhibitory activity by mouse B16-F10 cells.Conclusion:This research demonstrated that recombinant hTGF-?1can be expressed in both E.coli and Pichia yeast.Recombinant hTGF-?1 was successfully preparation in Escherichia coli expression system,and its purification and renaturation methods were established.It was demonstrated by cell experiments that the recombinant hTGF-?1 has melanin production inhibitory activity.