甘油脱水酶gldC基因克隆、表达与鉴定

Cloning and Expression of Glycerol Dehydratase gldC Gene

目的 :表达及纯化甘油脱水酶γ亚基蛋白。方法 :使用PCR及DNA重组技术 ,将甘油脱水酶γ亚基基因gldC重组到含麦芽糖结合蛋白 (MBP)的融合蛋白表达载体pMAL -c2x中 ,在大肠杆菌中进行表达。结果 :转化重组质粒pMAL gldC的大肠杆菌经IPTG诱导 ,SDS -PAGE分析显示表达出的MBP -gldC融合蛋白相对分子质量约 6 6kD ,与预期大小一致 ,并经Westernblot分析证实。用直链淀粉树脂亲和层析纯化得到电泳均一的融合蛋白。结论 :成功地获得甘油脱水酶γ亚基融合蛋白 。

Objective:To express and purify the protein subunit of glycerol dehydratase.Methods:PCR and DNA Recombination techniques were employed,gldC gene was cloned into express vectorpMAL-c2xand the fusion protein MBP-gldC was expressedin E.coli.Results:E.coli.DH5αcells withplasmid pMAL/gldC were induced by IPTG for 4 hours.SDS-PAGE analysis showed that the molecular weights of MBP-gldC was about 66kD.Western blot analysis showed fusion protein presented the specific MBP antigenicity.MBP-gldC fusion protein were purified by amylose resin affinity chromatography and showed to be homogeneity in SDS-PAGE.Conclusion:The MBP-gldC fusion protein was obtained and maybe suitable for studying biological functions and potential utility.