Construct Prokaryotic Expression Vector Of Human Collagen And Expression In E.coli

Collagen is a kind of biological macromolecules synthesis by animal fibroblasts,with supporting organs and protect the body function. With the development of biotechnology,people have a wide-ranging and profound understanding of collagen.Unique fibrous structure,good biocompatibility and low antigenicity determines that collagen have an important positionand promising application perspectivein in many areas. As an important natural protein resources, whether in biology and medicine, cosmetics, health food or the application in textile industry,has wide application prospects.But the traditional extraction method has such disadvantages as the water-insoluble, virus infectious, allograft tissue rejection.In recent years, with the genetic engineering, protein engineering science and technology development, using genetic engineering method to express purpose protein has become a hot topic.Objective:The Col6a2 gene was inserted into Prokaryotic expression vector pET-22b and expressed the fused gene in E. coli BL21AI (DE3) at a high level, in order to obtain a large number of soluble collagen.Method:Using method of RT-PCR, obtain the gene that encodes Collagen peptide,and digested it with restriction enzyme and ligated the insert to prokaryotic expression vector pET-22b.The recombinant plasmind was transformed into the E.coli BL21AI (DE3),identified position clones,sequencing analysis.Then induced to express by IPTG and confirmed the production by SDS-PAGE. Collecting the target protein by ultrasonication and centrifugation,then anasysis the solubility. The fusion protein was purified by Ni- NTA affinity chromatography What is more, on the base of small batch shake flask expression, we optimized the conditions of Col6a2 fermentation tank, which made a foundation of industry production.Result:The recombinant plasmid was transformed into E. coli BL21AI (DE3).The fusion protein expressed was about 46KDa and the target protein more existed in supernatant. Western blotting result indicated that the purified product was mature human collagen peptide; The high performance liquid chromatography (HPLC) showed that the purity of human collagen peptide was higher than 90%.The best expression conditions of the fusion protein was:the culture volume 50mL/500mL,inoculation volume 2% and incubated for 2.5h.Then 0.7 mmol/L IPTG was fed as inductor to the E.coli BL21(DE3)-pET-COL6A2 culture broth and maintained for 8h.The expression is up to 22.16%Conclusion:This study was able to promotes Col6a2 soluble expression of the latter in E. coli, making it more convenient to purify Col6a2 than previously. This may be a better method to produce collagen peptide for industry research and development.