大腹园蛛次壶腹腺丝的表达

Expression of Araneus ventricosus minor ampullate spidroin

摘要:

基于大腹园蛛次壶腹腺丝Minor Ampullate Spidroin全长编码基因最新报道，研究了该基因的表达。利用PCR扩增该基因重复区一段长1 348 bp的片段P1，融合his-tag标签，构建酵母表达载体，在毕赤酵母菌GS115进行表达。同时构建大肠杆菌表达载体，在大肠杆菌BL21(DE3) 中进行表达。SDS-PAGE和Western blotting检测结果表明，P1在两种表达系统中均可实现表达。研究结果显示：P1在GS115中的表达经优化后产量、产率有较大提高，且远高于BL21(DE3) 中的表达，相应的纯化效率GS115也远高于对照BL21(DE3) 的表达。研究表明酵母表达系统更适合重复度高、且富含Gly/Ala的天然蛛丝蛋白基因的表达，为表达全长天然MiSp编码序列提供前期实验基础，也为大规模蛛丝蛋白的重组表达建立了平台。

Abstract:

A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.