人CaMKKβ蛋白的原核表达、纯化及活性测定

Prokaryotic expression, purification and activity assay of human CaMKKβ protein

目的对原核体系表达的CaMKKβ蛋白体外活性进行探索,为针对CaMKKβ、AMPK药物研发提供科研基础。方法克隆人CaMKKβ基因,建立原核表达载体pET28a-CaMKKβ,将其转化入E.coli Rosetta(DE3)感受态细胞内,在16℃、0.02 mmol/L IPTG条件下诱导重组表达CaMKKβ蛋白,Ni-NTA纯化6His-CaMKKβ蛋白,并利用Glo?Max Assay方法检测其活性。结果通过基因测序表明pET28a-CaMKKβ质粒构建成功;重组人CaMKKβ蛋白可溶性表达量较高,Ni-NTA纯化6His-CaMKKβ蛋白纯度可达80%以上,活性检测结果表明,大肠杆菌体系内表达的人CaMKKβ蛋白在CaM/Ca2+存在与否情况下,酶活基本一致。结论成功构建原核表达载体pET28a-CaMKKβ,实现重组人CaMKKβ在原核体系内可溶性表达,活性测定表明原核体系内表达的CaMKKβ自主酶活性较强,受CaM/Ca2+影响较小。

Objective To facilitate drug discovery based on CaMKKβ and AMPK, cloning, expression and purification of human CaMKKβwere performed in Escherichia coli. Methods Human CaMKKβ gene was cloned to bacterial expression vector pET28a-CaMKKβ, and then transformed into E.coli Rosetta（DE3） cells. The recombinant CaMKKβprotein was induced with 0.02 mmol/L IPTG at 16 ℃, 6His-CaMKKβwas purified through Ni-NTA chromatography and activity was assessed by Glo？Max Assay. Results The expression vector pET28a-CaMKKβwas confirmed via DNA sequencing. After optimization of induction condition, recombinant human CaMKKβ protein was expressed at high level mainly as soluble form. Single step Ni-NTA chromatography generated high purity product （greater than 80%）. Activity analysis revealed that CaM/Ca2＋did not modulate the enzymatic activity of recominant CaMKKβ. Conclusions pET28a-CaMKKβvector is constructed successfully. Recombinant human CaMKKβis purificatied by Ni-NTA, and strong autonomous activity of recombinant CaMKKβexpressed in prokaryotic system is not affected by CaM/Ca2＋.