将南极嗜冷菌Moritella sp.2-5-10-1的低温脂肪酶基因Lip-837克隆到表达载体pCold Ⅲ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%.%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 pCold Ⅲ. The recombinant plasmid pCold Ⅲ +Lip-837 is constructed, and then transformed into E. coli BL21 (DE3). It is shown in the results from the SDSPAGE 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 PTfl6) 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 pCold Ⅲ +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.
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