重组羧肽酶原B的复性方法研究

构建的羧肽酶原B表达质粒在大肠杆菌中获得高表达。但目的蛋白是以包涵体的形式存在。为了获得活性羧肽酶B,必须对其包涵体进行变复性。首先利用稀释复性确定了羧肽酶原B复性的最佳缓冲液;在凝胶过滤复性中,研究了柱长和洗脱流速对羧肽酶原B复性效率的影响;另外对比了稀释复性、透析复性、凝胶层析复性和Ni2 亲合层析法等四种方法对羧肽酶原B的复性效果。结果发现,这4种方法的复性效果有以下顺序:凝胶过滤复性>稀释复性>Ni2 亲合层析>透析复性。

Study on Refolding Ways of Recombinant pro-Carboxypeptidase B

Recombinant pro-carboxypeptidase B forms insoluble inclusion bodies when overexpressed in Escherichia coli, and it must be denatured and renatured in vitro before it acquires activity. Firstly, we optimized the renaturation buffer by dilution ways. In the urea gradient gel filtration, we studied the column length and elution velocity on pro-carboxypeptidase B refolding effect. Finally, refolding effects of four ways including dilution, dialysis, urea gradient gel filtration and Ni 2 immobilize metal affinity chromatography were comparaed. The results indicated that the sequence of refolding effect was as follows: urea gradient gel filtration>dilution> Ni 2 immobilize metal affinity chromatography>dialysis.