MMP9-PEX的原核表达、纯化与复性研究

目的 对人基质蛋白酶9（matrix metalloproteinase,MMP9）C端血红素结合蛋白样结构域（hemopexin domain,PEX）的原核表达条件、透析复性条件进行优化,以获得高表达的活性蛋白.方法 将构建好的原核表达载体PET-his-MMP9-PEX转化至大肠埃希菌菌株BL21（DE-3）,异丙基β-D硫代半乳糖苷（IPTG）诱导后产生包涵体蛋白,探讨不同IPTG浓度、不同诱导时间、不同菌液密度值对目的 蛋白表达产量的影响;盐酸胍裂解包涵体,采用NI-NTA进行纯化,对纯化蛋白的透析复性条件进行优化;采用明胶酶谱的方法检测MMP9-PEX的活性.结果 经纯化透析复性后蛋白的纯度在95%以上,复性回收率为36.86%.结论 本研究优化了MMP9-PEX的原核表达、透析复性条件,获得了有活性的MMP9-PEX蛋白,为进一步研究MMP9-PEX的功能提供了实验基础.

Prokaryotic Expression and Purification and Renaturation of MMP9-PEX

Objective To optimize conditions for prokaryotic expression, purification and renaturation of human carboxyl-terminal hemopexin domain （PEX） of matrix metalloproteinase （MMP9） . Methods The expression vector, PET-his-MMP9-PEX, was transformed into E. coli BI21 （DE3） under IPTG induction. Expression level of PEX was examined in various culture conditions, including IPTG concentration, induced time, OD values of bacilli-solution and renatured conditions with various dialysis. Cytoplasmic inclusions were cleaved by guanidine hydroehloride. The recombinant protein was purified by affinity chromatography. Enzymatic activity of MMP-9-PEX was detected by gelatin zymography. Results The purity of renatured MMP-9-PEX protein was a- bove 95% , with36. 86% of renaturation rate. Conclusion The conditions for prokaryotic expression, purification and renaturation of functional human MMPg-PEX were optimized, providing experimental basis for further study of MMP9-PEX function.