MMP-9-PEX原核表达、纯化及其对结肠癌细胞侵袭能力的影响

用RT-PCR法扩增出基质金属蛋白酶-9(MMP-9)C末端血红素结合蛋白样结构域(PEX),克隆至表达载体pET32a中并转化至E.coilBL21(DE3),经IPTG诱导后在约为42kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量占菌体总蛋白的50%左右。重组蛋白质pET32a/PEX主要以包涵体形式表达,其在上清液中的含量极微。含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包涵体采用金属整合层析有效分离出目标蛋白,纯化后pET32a/PEX蛋白质的纯度大于90%。在Transwell实验中发现复性纯化后的重组蛋白质pET32a/PEX能抑制结肠癌细胞的侵袭,且呈剂量依赖性。

Expression, Purification of the MMP-9-PEX in E. coil and Its Effect on Invasive Capacity of Colonic Cancer Cells

Matrix metalloproteinase 9 hemopexin domain(PEX) ,which locates the C-terminal,wasamplified by RT-PCR and was inserted into expression vector pET32a.The recombinant plasmid was induced byIPTG for 4 h and a 42 kDa recombinant protein was produced.Amount of the fusion protein pET32a/PEX expres-sion was 50% of total bacterial protein,mostly in form of inclusion,few in form of supernatant.Inclusion wasdissolved in 8 mol/L urea and 10 mmol/L DTT,carried out affinity purification under denaturing condition.Theexpressed 42 kDa fusion protein is confirmed by SDS-PAGE.The pET32a/PEX expression vector was successfullyconstructed and highly expressed in E.coli.The purified pET32a/PEX protein can inhibitor the invasion of thecolonic cancer cell in Transwell experiments and show a dose-dependent manner.