【摘要】 将南极嗜冷菌Moritellasp.2-5-10-1的低温脂肪酶基因Lip-837克隆到表达载体pColdⅢ中,成功构建了重组质粒pColdⅢ+Lip-837,并转化大肠杆菌E.coli BL21(DE3)。SDS-PAGE实验结果表明,实现了该基因在E.coli的异源表达,且其表达量达到细胞总蛋白的39%;但表达产物主要以包涵体形式存在。将该重组质粒pColdⅢ+Lip-837分别与不同分子伴侣质粒,如pGro7,pKJE7,pG-Tf2,pTf16在E.coli BL21(DE3)中实现了共表达。SDS-PAGE结果表明,4种分子伴侣均能明显提高目的蛋白—脂肪酶Lip-837的可溶性表达,但不同分子伴侣提高可溶性蛋白表达的程度有所不同,如仅表达pColdⅢ+Lip-837时,低温脂肪酶可溶性蛋白占总表达量的比例为5%;当与分子伴侣pGro7共表达时,对脂肪酶可溶性蛋白表达提高的比例最大,其可溶性酶蛋白的比例可占总表达量的42%;分子伴侣质粒pG-Tf2促进可溶性表达的效果较差,其可溶性酶蛋白的比例仅占其总表达量的16.6%。更多还原

【Abstract】 The cold-active lipase gene Lip-837 is cloned from Antarctic psychrophilic bacterium Moritella sp.2-5-10-1 and ligated into the expression vector pColdⅢ.The recombinant plasmid pColdⅢ+Lip-837 is constructed,and then transformed into E.coli BL21(DE3).It is shown in the results from the SDS-PAGE experiment and analysis that substantive heterologous expression of Lip-837 protein into E.coli is realized,and due to the heterologous expression the recombinant protein,mostly in the forms of the inclusion bodies,can be as much as 39% of the total protein contatined in the bacterial cells.In addition,the co-expressions of the recombinant plasmid pCold Ⅲ+Lip-837 respectively with the molecular chaperiones(pGr07,pKJE7,pG-Tf2,and PTf16) are also achieved.As the results from SDS-PAGE experiments show,in some degree the various chaperones are significantly improve the soluble protein expression of the Lip-837 lipase,i.e.the expected protein.The soluble protein in the cold-active lipase is as much as 5% of the total expressed protein in the experiment for the pCold Ⅲ+Lip837 expression without any chaperone.It becomes 42% in the experiment for the co-expression of pColdⅢ+Lip-837 with pGr07,the highest among the experiments of the co-expressions respectively with the 4 chaperones.The effect of the solubility expression with pG-Tf2 becomes the least significant,and the soluble protein is as much as 16.6% of the total expressed protein.更多还原