人骨钙素真核表达载体构建及蛋白表达纯化

Construction of secretory expression vector of BGP and expression and purification of BGP protein

[目的]在毕赤酵母真核表达系统中获得人骨钙素(h BGP)蛋白以便用于2型糖尿病新药研究。[方法]人工合成h BGP基因并克隆入真核载体p PIC9K,转化毕赤酵母GS115,0.5%甲醇诱导表达,优化表达时间,实现h BGP的真核表达。获得的目的蛋白经离子交换层析和分子筛等进行纯化,用Tricine-SDS-PAGE检测蛋白表达情况及纯化结果,并以Western blot进行验证。[结果]酶切及基因测序结果显示成功构建了p PIC9K-BGP真核表达载体,经Tricine-SDS-PAGE及Western blot测定均检测到分子量为5.8k Da的目的条带,表明h BGP在毕赤酵母中成功表达并纯化。[结论]构建的p PIC9K-BGP真核表达载体能在毕赤酵母中成功表达分子量为5.8k Da的h BGP蛋白,经纯化后目的蛋白纯度在95%以上。

[Objective] The human bone γ -caboxyglutamic acid- containing protein （hBGP） in Pichia pastoris was ex- pressed for development of new drug for type 2 diabetest.[ Methods ] The gene sequence of hBGP was obtained by gene synthe- sis to construct pPIC9K- BGP secretory expression vector and transfect into Pichia pastoris GS115 and induced to express by methanol, and meanwhile the expression condition was optimized to obtain thesecretory expression of hBGP. The hBGP was puri- fied with ion - exchange column chromatography and molecular sieve filtration, and deteced by Tricine - SDS - PAGE and west- ern blot analysis. [ Results] The results of enzyme digestion and DNA sequencing indicated that pPIC9K -BGP was successful- ly constructed , and hBGP was expressed and purfied in Pichia pastoris successfully by Tricine - SDS - PAGE and Western blot analysis which showed the 5.8 kDa target protein . [ Conclusion ] hBGP of 5.8 kDa molecular weight was successfully ex- pressed in Pichia pastoris by the secretory expression vector pPIC9K - BGP and the protein purity quotient was more than 95% after purification.