水稻巯基蛋白酶抑制剂cDNA在大肠杆菌中的表达

Expression of Oryzacystatin in Escherichia coli

根据从水稻未成熟种子cDNA文库中筛选出的水稻巯基蛋白酶抑制剂(Oryzacys-tatin)cDNA序列,利用聚合酶链反应(PCR)技术扩增出Oryzacystatin编码区,插入到温度敏感型大肠杆菌表达载体的P_RP_L启动子下游,该质粒带有温度敏感型阻遏子的编码基因cIts857。转化大肠杆菌DH5α后,通过升温诱导,Oryzacystatin在大肠杆菌中获得高效表达,SDS-PAGE表明分子量约为12kDa,与预期结果一致,表达量占细菌可溶性蛋白总量的10％以上;对巯基蛋白酶的抑制活性检测表明,可溶性蛋白组分对木瓜蛋白酶有明显的抑制活力。

A rice cDNA library of immature seeds had already been constructed, from which colonies carrying oryzacystatin cDNA were isolated. Coding region of the oyzacys-tatin was amplified by polymerase chain reaction (PCR) and inserted downstream of PRP1. promoter of E. coli thermal-inducible expression vector pBV220. An oryzacystatin expression plasmid pBVG9 was thus obtained. Shifting the culture temperature of E. coli DH5a (pBVC9) from 30C to 42C led to a high level expression of oryzacystatin. The result of SDS-PAGE showed a distinct band of 12.0kDa which accounts for at least 10 % of the total soluble proteins. The inhibitory activity of the total soluble proteins of E. coli DH5a (pBVC9) toward papain was evaluated by using that of E. coli DH5a (pBV220) as a control. Results evidently indicated that soluble proteins of E. coli DH5a (pBVC9) exhibited inhibitory activity toward papain.