高分子量RGD-蛛丝蛋白重组体的构建、高密度发酵及纯化

Construction，Fermentation and Purification of High Polymer Spider Dragline Silk Protein Containing RGD Peptide

摘要:

蜘蛛丝是自然界综合性能优良的天然蛋白质纤维之一，因其具有良好的生物相容性和可降解性在生物医学领域具有潜在的应用前景。 在本室已经构建的RGD-蜘蛛拖丝蛋白基因16多聚体基础上，通过首尾相连、倍加等方法进一步多聚化，得到RGD-蜘蛛拖丝蛋白基因32和64多聚体，分别将这两种多聚体与原核高效表达载体pET-30a(+)连接，转化大肠杆菌BL21(DE3)pLysS，得到的32多聚体表达重组子命名为pNSR32，64多聚体表达重组子命名为pNSR64。通过酶切、琼脂糖电泳鉴定及对目的片段的测序均与理论值相符。将32和64多聚体基因序列注册GenBank，序列号分别为DQ469929和DQ837297。重组体pNSR32和pNSR64经IPTG诱导表达，SDS-PAGE图谱显示表达产物分子量分别为102kD和196.6kD， 与天然蛛丝蛋白分子量接近并与理论值相吻合。高分子量的蛛丝蛋白在原核生物成功实现高效表达，在国内外尚未见报道。在此基础上对pNSR32工程菌进行高密度发酵，建立了简单高效的目的蛋白纯化工艺。

Abstract:

Spider silk is a natural protein fibroin with excellent character as it is light and tenacious. It has a wild potential applications in the biomedical field due to its good biocompatibility and degradation. Arginine-glycine-aspartic acid (RGD) is a highly conserved amino acid sequence of many adhesion protein. Biological materials binding with RGD peptide in the surface can promote cells adhesion， migration and proliferation.Our lab had constructed the 16 muhimers with the introduced RGD peptide codons which involve cell adhesion for the first time. It was found that the mechanical capability of the 16 mulimer protein was very limited because of the big gap in molecular weight with nature spider proteins when it was used to made biomaterial scaffold.In this paper,based on the 16 multimers of the highly,repetitive sequence of spider dragline silk and with RGD peptide condons which has been constructed by our lab forestall,it was used to construct the 32 and 64 multimers sequence of spider dragline silk by the strategy of “head to tail". The 32 and 64 multimers were ligated into prokaryotic expression vector pET-30a，and then the Bl21(DE3)pLysS. The fragments were in agreement with the desired through digestion,agarose gel electrophoresis respectively.By registration into the GenBank data-base,the serial numbers of DQ469929 and DQ837297 were gained respectively. The expression of recombinant protein was introduced by the addition of IPTG. SDS-PAGE analysis shows that the molecular weight of products expressed here are 102kD and 196.6kD in agreement with the desired respectively. It was the first time for the high polymer spider dragline silk protein expressed in prokaryotic biology. Furthermore，a larger quantity of synthetical proteins with high density fermentation were searched after,and a suit of high efficient purification methods for 32 multimers protein were established.