Prokaryotic Expression,Purification and Enzymatic Characterization of Recombinant Human Phospholipase A₂

The prokaryotic expression and purification of human phospholipase A2 was realized,purification and the enzymatic characterization was analyzed. Human PLA₂(PLA2G10)was identified by searching NCBI,and constructed on the vector pET28a+,and maltose binding protein(MBP)was used as the solubilizing label. The vector pET28a-MBP-PLA was successfully constructed. After purification BL21(DE3)was transformed,it was induced to express at low temperature,and identified by SDS-PAGE,and confirmed to be massively expressed in the supernatant. According to the properties of the protein,a set of protein purification process was established,including Q-column elution,ammol/Lonium sulfate salting out,phynyl column purification,and xylose column purification. The purification of the recombinant enzyme was more than 90% by SDS-PAGE. The specific activity of lecithin was 127.04 U/mg by acid-base titration. The purification yield was 48.8%,and the purification multiple was 10.3 times. The study of enzymatic properties showed that the enzyme had a molecular weight of 56.5 kDa,the optimum reaction temperature was 40 ℃,the optimum reaction pH was 8.0.It was a calcium dependent enzyme with the strongest affinity to PC,K_m value of 12.2 mmol/L,V_max of 0.19 mmol/L/min. In this study,the heterologous expression and purification system of phospholipase A₂ was established,which laid a foundation for further theoretical and industrial research.