心肌特异表达基因p93的激酶及丝氨酸结构域在大肠杆菌中的高效表达  

High level expression of kinase and serine domain of cardiac-specific gene p93 in Escherichia coli

作　　者：尤昕[1] 时娜[1] 柳明洙[2] 曹慧青[1] 刘冬青[1] 孟宪敏[1] 

机构地区：[1]中国医学科学院中国协和医科大学阜外心血管病医院分子医学中心 [2]延边大学医学院生物化学与分子生物学教研室,延吉市133000

出　　处：《中国分子心脏病学杂志》2004年第6期330-333,共4页Molecular Cardiology of China

基　　金：国家自然科学基金资助项目 (No 30 2 70 557);北京市重大科技项目 (No H0 2 0 2 2 0 0 2 0 31 0 )

摘　　要：目的　在大肠杆菌中高水平表达并分离纯化心脏特异表达基因p93的激酶与丝氨酸结构域片段。方法　利用DNA重组技术 ,构建C末端带有 6×His标签的p93激酶与丝氨酸结构域(p93 C)片段的原核表达载体pET2 8a p93 C。对诱导表达条件和包涵体洗涤条件优化后 ,用Ni2 +金属螯合吸附层析柱纯化。结果　成功构建了pET2 8a p93 C原核表达载体 ,诱导后摇床转速在 35 0r/min下表达量最高 ,包涵体洗涤在超声条件下较好 ,分离纯化后从 10 0ml菌液中可获得 2 5mg蛋白。结论建立了高效稳定的p93 C原核表达体系以及较好的包涵体洗涤方法。Objective To highly level express and purify the fragment of kinase and serine domain of cardiac-specific gene p93 in Escherichia coli. Methods Using DNA recombinant technology,the fragment of kinase and serine domain of cardiac-specific gene p93 with C-terminal 6 × His tag was inserted pET28a （ ＋ ）. The fusion protein was purified with Ni^2 ＋ metal chelate affinity chromatography columns, after optimization of expression level and inclusion body washing conditions. Results Recombinant expressing vector pET28a- p93-C was successfully constructed, high level expression was gained with 350 r/min shaking for 2-3h at 37℃ after inducing, good washing result of inclusion body was achieved with ultrasonicate. 2.5mg of p93-C was obtained from lOOml of E. coli. Conclusion The high level expression and good inclusion body washing methods were set up.

关 键 词：心脏特异基因p93 原核表达 分离纯化 

分 类 号：R541[医药卫生—心血管疾病]