精氨酸激酶C端结构域的克隆及其表达纯化  

Cloning,Expression and Purification of the C-Domain of Arginine Kinase

作　　者：雷婧[1] 万华涛 汪劲松[1] 王卫东[1] 潘继承[1] 

机构地区：[1]湖北师范学院生命科学学院蛋白质结构与功能实验室,湖北黄石435002 [2]湖北省黄石广播电视大学阳新分校,湖北黄石435200

出　　处：《化学与生物工程》2010年第6期41-44,共4页Chemistry & Bioengineering

基　　金：国家自然科学基金资助项目(30570412);湖北省自然科学基金资助项目(2005ABA192)

摘　　要：精氨酸激酶（Arginine kinase,AK）由一个小的球形N端结构域和一个大的C端结构域构成。将来源于基围虾的AK C端结构域基因成功克隆到了原核表达载体pET-28a上,然后再转入Rosetta进行表达。探讨了AK C端结构域的最佳表达条件,当OD600达到0.5~0.7时,用终浓度为0.2 mmol.L-1的异丙基硫代--βD-半乳糖苷（IPTG）在20℃下诱导培养5~6 h,AK C端结构域在上清中大量表达。采用His-Tag金属螯合亲和层析纯化、不连续梯度咪唑洗脱,得到了电泳纯的AK C端结构域。Arginine kinase（AK） plays an important role in cellular energy metabolism in invertebrates.AK consists of a small spherical N-terminal domain and a large C-terminal domain.The encoding AK gene from greasyback shrimp was cloned in prokaryotic expression plasmid pET-28a,and then it was expressed in Rosetta dissoluble form.It was found that AK C-domain was expressed in bulk in supernant when the culture induced by isopropyl-β-D-galatose with final concentration of 0.2 mmol·L-1 lasted for 5~6 h at 20℃ when OD600 was 0.5~0.7.The electrophoretic pure recombinant protein was obtained by purification using His-Tag nickel affinity chromatography and discontinuous gradient elution with imidazole.

关 键 词：精氨酸激酶 C端结构域 克隆 表达 纯化 

分 类 号：Q786[生物学—分子生物学]