Purification And Wet-spinning Of Recombinant Spider Silk Protein Produced By High Cell Density Fermentation

Spiders produce a variety of high-performance structural fibers including the major ampullate dragline silk with remarkable mechanical properties combining tensile strengh, toughness and light weight. 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 silk protein. The purpose of this research was to establish technique to produce recombinant spider silk protein through high cell density fermentation and simple large-scale purification method, and artificially synthesize fibers by wet spinning. Here described is the first known report of spinning recombinant spider silk fibers domestically.Two engineering bacterial strains were used to express the vector encoding recombinant spider silk protein in shaking bottle and 10 L fermentor cultivation. It showed BL21(DE3) was the better bacterial host. The method of pH-stat fed-batch culture was applied to obtain high cell density. The final cell density OD600 of grown pNS2/BL21(DE3) was 71.64 and the volumetric productivity was 0.8g/L.The recombinant protein was expressed as a soluble product in E.coli. The iso-electric point was 8.5. A series of purification steps, including cell breakage by sonication, regulating solution pH5.6, 55~60C heating for 4min, and 20%(NH4)2SO4 precipitation. The final protein purity was 92% and the recovery was 57.5%.To prepare spin dope, formic acid was used to dissolve the dialyzed and lyophilized recombinant protein achieving a protein concentration more than 10%. Factors which affect the wet spinning was partly studied. Some properties of three formed fiber synthesized in different wet spinning conditions was analyzed including solubility in water, density and tenacity. Fibers insoluble inwater with certain tenacity was synthesized. Fiber formation, appearance and properties at least depended on spin dope concentration, coagulation bath composition and concentration, and post-spinning draw. It was anticipated that by optimization of spinning parameters, genetic engineered silk fibers with higher properties could be produced.