人腺苷激酶在大肠杆菌中的表达与纯化

Prokaryotic Expression and Purification of Human Adenosine Kinase

目的诱导人腺苷激酶重组蛋白在大肠杆菌中表达,并对表达的重组蛋白进行纯化。方法将由人腺苷激酶基因开放阅读框与原核表达载体pET30a(+)连接构建的重组蛋白表达载体pET30a(+)/ADK转化大肠杆菌表达菌株BL21(DE3)感受态细胞。1mmol/L的IPTG诱导重组蛋白表达。离心收集菌体后结合使用溶菌酶、冻融和超声破碎的方法裂解菌体。用含2mol/L和4mol/L尿素的结合缓冲液洗涤包涵体去除部分杂蛋白。随后用含8mol/L尿素的结合缓冲液变性溶解剩余包涵体沉淀。用Ni-NTA柱通过亲和层析纯化重组蛋白,用含8mol/L尿素的洗脱缓冲液洗脱,收集样品透析复性。结果成功构建人腺苷激酶原核表达载体且目的蛋白以包涵体形式表达。经包涵体洗涤、变性溶解,亲和层析及分步透析复性获得纯度达85%以上的重组蛋白。结论人腺苷激酶表达载体的成功构建以及重组蛋白的纯化,为进一步进行其结构和功能的研究奠定了基础。

Objective To induce human adenosine kiuase recombinant protein expression in E.coli and purify the recombinant protein. Methods The human adenosine kinase recombinant protein expression vector pET30a（＋）/ ADK was constructed by connecting the ORF of human adenosine kinase gene and prokaryotic expression vector pET30a（＋ ）. The recombinant vector was transformed into E.coli BL21 （DE3）. The recombinant protein was induced by adding IPTG in culture medium to 1 mmol/L. The cell pellet were lysed by the combined use of sonication and lysozyme. The pellet of inclusion bodies was washed with binding buffer which containing 2 mol/L and 4 mol/L urea and dissolved with binding buffer containing 8 mol/L urea. The supernatant was used for affinity chromatography using Ni-NTA column, The recombinant protein sample was obtained by eluted Ni-NTA column with elution buffer which containing 8 mol/L urea. SDS-PAGE gel eleetrophoresis was carried out to analyze the specificity of recombinant protein. Results Human adenosine kinase recombinant protein expression vector was successfully constructed by seamless cloning method. The recombinant protein was expressed as inclusion bodies and its molecular weight was about 46 kDa, consistent with the expecting value. Through affinity chromatography recombinant protein was purified and the purity coefficient was up to 85%. Conclusion Highly purified human adenosine kinase recombinant protein was obtained and can be used directly to prepare antibody and may facilitate further protein role and structure investigation.