微小毛霉凝乳酶基因的克隆及在毕赤酵母GS115中的表达

以微小毛霉基因组为模板,PCR扩增得到凝乳酶结构基因,经EcoRⅠ和NotⅠ双酶切后,连接穿梭载体pPIC9K,转化大肠杆菌感受态细胞DH5α,并测定其核苷酸序列。测序结果证实扩增得到的片段为凝乳酶基因,与GenBank报道的序列（No.X06219）存在6对碱基的区别。将重组质粒电转化毕赤酵母GS115,构建重组菌P.pastorisGS115/pPIC9K-m cp,通过筛选得到抗G418浓度达到3.0 mg/m l、具有多拷贝基因的整合重组菌pPIC9K-m cp-03。用甲醇诱导进行初步发酵试验,测得该重组菌凝乳酶活力为5 660 U/m l,比微小毛霉发酵液提高50多倍,且发酵液电泳结果表明毕赤酵母自身分泌的蛋白很少,只能看到约38.5 ku的凝乳酶蛋白条带。

Cloning of Mucor pusillus Rennin Gene and Its Expression in Pichia pastoris GS115

The rennin gene was amplified from Mucor pusillus by PCR and was recombined into a shuttle vector pPIC9K after digestion by EcoRⅠ and Not Ⅰ.Then a plasmid of pPIC9K-mcp was constructed through transformation into E.coli DH5α,and the nucleic acid sequence encoded by the gene was sequenced.The results showed that the amplified fragment was the rennin gene with six pairs of base different from the GenBank sequence No.X06219.The recombination P.pastoris GS115/PIC9K-mcp was constructed by electroporating the vector into Pichia pastoris GS115.Through screening,the multi-copy recombination PIC9K-mcp-03 was obtained resistant to 3.0 mg/ml G418.By the initial ferment experiment induced by methanol,the rennin activity of PIC9K-mcp-03 was determined to be 5 660 U/ml,which was 50 times higher than that of Mucor pusillus.Moreover,less protein bands from Pichia pastoris appeared,as a result,only about 40 ku rennin protein bands were observed.