Studies On High Density Fermentation Of Recombinant Spider Silk Protein In E. Coli PNS2/BL21(DE3) And Optimization Of Purification And Histocompatibility Of Product

Spider silk is a special fibrin. It has high strength, elasticity, flexility, elongation and tensile strength, along with lightness, the endurance of ultraviolet radiation, biodegradation, and so on. As a biomaterial, spider silks have a good prospect of application. Advances in genetic engineering have taken great strides toward the production of large quantities of recombinant spider silk protein. The purpose of this research was to establish technique to produce recombinant spider silk protein in engineering E. coli pNS2/BL21(DE3) which was constructed by our laboratory through high cell density fermentation and simple large-scale purification method. Furthermore we discuss the histocompatibility of scaffold biomaterial constructed from purification product.We first report the optimization conditions of recombinant spider silk protein in engineering E. coli pNS2/BL21(DE3) by high cell density fermentation orthogonal experimental design. As a result of 9 times orthogonal experimental, the significant factors which effect expression of spider silk protein are ordinal pH, concentration of Gly/Ala,temperature and concentration of IPTG. The best condition of expression in high cell density fermentation is pH7.0, 32℃, 0.1% (w/v) Gly/Ala and 0.2mmol IPTG/L. We repeat the experiment, the final OD₆₀₀ is 55～60, expression rate is 22～24%.We optimize the purification method of recombinant spider silk protein, establish the technique of simple and high efficiency large-scale purification. The project was enhancing temperature and delaying the time of heat, and the efficiency to remove mixed protein was superior than former method. The final protein purity was above 85% and the recovery was 10mg/g through improve purification efficiency and protein harvest rate. The recovery was 3 times than former method, and the time reduce a half.We compare the histocompatibility of four scaffold biomaterials: spider silk protein pNS2, pNSR16, PVA and pNSR-Z by implant into mouse in vivo. The excellent sequence was pNSR16＞pNS2＞PVA＞pNSR-Z. It was showed that the scaffold biomaterials of pNSR16 and pNS2 was good tissue compatibility, it will be a good prospect of application.