蛛丝蛋白基因在大肠杆菌中表达研究

Expression of natural fragment of spider flagelliform silk protein in Escherichia coli

利用大腹园蛛基因组文库筛选获得一段693 bp基因片段,经分析该段基因处于鞭毛状丝基因重复区域,且其中包含了一个完整的重复框架。通过基因密码子优化,在其3′和5′端分别融合蛋白质亲和层析标签,克隆于pET30LIC表达载体中,在不同的大肠杆菌中进行表达试验。实验结果显示：经过密码子优化,融合目的蛋白基因在BL21（DE3）中得到了高效表达,产量达到25-30mg/L,纯化产物纯度达90%以上。SDS-PAGE和W estern-b lotting检测目的融合蛋白均与预期蛋白大小一致。

Industrial production of spider silks may best be achieved through the production of silk proteins in microorganisms.A 693-bp gene fragment encoding the repeat domain of the flagelliform silk was isolated from Araneus ventricosus gene library.Then this spider silk gene fragment was inserted into an expression plasmid and compared efficiency of the gene expression in different E.coli strains before and after codon-optimization.It revealed that the optimized gene could be expressed efficiently in E.coli strain BL21（DE3）.The resulting recombinant protein had a yield of approximately 25~30mg/L.It was purified to over 90% purity,and was identified by size and antibody recognition through SDS-PAGE and Western blotting.