人穿孔素氨基端肽段的原核表达与纯化

Expression and purification of recombinant N-terminal peptide of human perforin

目的 :用基因工程方法表达人穿孔素 (PFP)氨基端肽段 ,并加以纯化。 方法 :构建表达人 PFP蛋白 N端118个氨基酸肽段 (h PFP- N)的重组质粒 ,转化 E. coli BL 2 1菌株 ,经异丙基 -β- D-硫代半乳糖苷 (IPTG)诱导表达。在十二烷基肌氨酸钠存在下以超声处理 ,使得到的融合蛋白包涵体溶解。通过谷胱甘肽琼脂糖柱亲和层析获得纯化的 GST/h PFP- N融合蛋白 ,经凝血酶酶切去除 GST部分。 结果与结论 :p GEX- KG质粒能够高效表达 GST/h PFP- N融合蛋白 ,经谷胱甘肽琼脂糖亲和层析和凝血酶酶切后得到纯化的 h PFP- N蛋白。

Objectives: To express and purify N terminal peptide of human perforin(hPFP N). Methods: The recombinant expressive plasmid pGEX KG/hPFP N was constructed and then introduced into a strain of E. coli BL21(DE3). Upon the induction of IPTG, GST/hPFP N fusion protein was expressed. The expressed fusion protein was localized in inclusion bodies which could be solubilized by sonication after detergent lauroylsarcosine was added. The fusion protein was purified by affinity chromatography with glutathione agarose. After being cleaved by thrombin, GST and uncleaved fusion protein were removed by glutathione agarose beads once more, then purified recombinant rhPFP N protein was obtained. Results and Conclusions: GST/hPFP N fusion protein can be effectively expressed in E. coli and the protein hPFP N was obtained after the purification process.