HIV-1 Gag蛋白在大肠杆菌表达系统中诱导和纯化条件的优化

为探讨诱导温度对于HIV-1 Gag在大肠杆菌中表达产物状态以及尿素浓度对蛋白纯化效果的影响, 将30oC和37oC诱导表达的包涵体分别溶于不同浓度的尿素, 比较溶解性的差异, 并比较复性的不同。将30oC诱导的目的蛋白分别用2 mol/L和8 mol/L尿素溶解后做层析分离, 比较两者的分离效果。结果发现, 与37oC相比, 30oC诱导表达的蛋白能有效溶于低浓度尿素, 并且更容易复性。与8 mol/L尿素溶解相比, 30oC诱导的包涵体用2 mol/L尿素溶解后通过凝胶过滤和离子交换层析纯化能得到更好的分离效果。这提示低温诱导的Gag包涵体中可能含有更多类似天然态构象的蛋白, 而低浓度尿素有利于保持包涵体中蛋白的天然态构象。从而为包涵体蛋白的诱导表达和分离纯化提供了参考。

Optimization of Induction and Purification of HIV-1 Gag Protein in Escherichia coli Expression System

To investigate the effects of induction temperature on the expression product and the impact of urea concentration on the purification, HIV-1 Gag inclusion bodies from E. coli induced at 30oC (IB30) and 37oC (IB37) were dissolved with urea of different concentrations. The solubility and yield of refolding were compared. IB30 were dissolved with 2 mol/L and 8 mol/L urea, and then purified with chromatography. IB30 were found easier to be solubilized in low concentration of urea and easier to be refolded than IB37. Furthermore, compared to the IB30 dissolved in 8 mol/L urea, Gag protein solubilized in 2 mol/L urea was purified to higher purity with gel filtration (GF) and ion exchange (IEX) chromatography. Gag inclusion body induced at lower temperature may contain more protein with native-like or reversibly-denatured structures, and solubilization in the presence of low concentrations of urea can help to retain these structures. This study has provided new insights into the purification of proteins from inclusion bodies.