橡胶树3个可溶性无机焦磷酸酶基因的原核表达

摘要

Pyrophosphatase plays an important role in the regulation of the rubber biosynthesis of H. brasiliensis. In this work, the coding sequences of three soluble inorganic pyrophosphatase genes previously cloned by the authors were inserted into pGEX-6P-l of the E. coli expression vector. The expression vectors of pGEX-HbSIPl, pGEX-HbSIP2 and pGEX-HbSIP3 were transformed into E. coli and inducted expression. The result showed that transformants harboring the expression vectors of pGEX -HbSIPl and pGEX -HbSIP2 revealed a high level expression of fusion proteins in the E. coli BL21(DE3), but pGEX-HbSIP2 only expressed in the E. coli Rosetta (DE3)supplementing rare codons, the expression rate was 35.4%, 43.7% and 18.5% respectively. Recombinant proteins of HbSIPl-GST, HbSIP2-GST and HbSIP3-GST were purified. The total recovery of fusion protein was about 7%, 8% and 3%, respectively. This result provided a solid foundation for further research on the molecular characters, antibody preparation and functional studies of the three pyrophosphatases.%焦磷酸酶对橡胶的生物合成具有重要的调控作用.将已克隆的3个可溶性无机焦磷酸酶基因的编码区插入到原核表达载体pGEX-6P-1上,构建3个焦磷酸酶基因表达载体(pGEX-HbSIPl、pGEX-HbSIP2和pGEX-HbSIP3),再将表达载体转人大肠杆菌表达菌株进行诱导表达.结果表明:pGEX-HbSIP1、pGEX-HbSIP2表达载体在大肠杆菌BL21(DE3)中获得高效表达,pGEX-HbSIP3只在补充稀有密码子的大肠杆菌Rosetta (DE3)中表达,表达率分别为35.4％、43.7％和18.5％.对原核表达的蛋白质进行纯化,获得3种可溶性重组蛋白GST-HbSIP1、GST-HbSIP2和GST-HbSIP3,总回收率分别约为7％、8％、3％.该结果为进一步研究这3种焦磷酸酶蛋白的分子特性、抗体制备及功能研究奠定基础.