Prokaryotic Expression and Purification of Human Adenosine Kinase

Objective To induce human adenosine kinase 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 electrophoresis 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.