CAJ | 학술논문

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。
목적대원핵체계표체적 CaMKKβ단백체외활성진행탐색，위침대 CaMKKβ、AMPK 약물연발제공과연기출。 　　방법극륭인 CaMKKβ기인，건립원핵표체재체pET28a-CaMKKβ，장기전화입 E.coli Rosetta(DE3)감수태세포내，재16℃、0.02 mmol/L IPTG 조건하유도중조표체CaMKKβ단백，Ni-NTA 순화6His-CaMKKβ단백，병이용 Glo?Max Assay 방법검측기활성。 　　결과통과기인측서표명 pET28a-CaMKKβ질립구건성공；중조인 CaMKKβ단백가용성표체량교고，Ni-NTA 순화6His-CaMKKβ단백순도가체80%이상，활성검측결과표명，대장간균체계내표체적인 CaMKKβ단백재 CaM/Ca2+존재여부정황하，매활기본일치。 　　결론성공구건원핵표체재체 pET28a-CaMKKβ，실현중조인 CaMKKβ재원핵체계내가용성표체，활성측정표명원핵체계내표체적 CaMKKβ자주매활성교강，수 CaM/Ca2+영향교소。
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+.