摘要
[目的]体外表达并纯化具有活性的CtaP蛋白,体外研究其与GSH的结合能力。[方法]利用PCR技术扩增CtaP基因,克隆到p Waldo-GFPe质粒构建重组子p Waldo-CtaP,转化大肠杆菌BL21(DE3)。通过优化IPTG浓度和诱导温度,经镍柱亲和层析和分子筛层析纯化获得可溶且稳定的CtaP蛋白。体外检测CtaP与底物GSH的结合能力。[结果]成功构建p Waldo-CtaP表达重组子,于18℃、0.1 mmol/L IPTG诱导培养20 h,蛋白质表达量较高且主要以可溶的形式存在。Native-PAGE显示CtaP能够与GSH或GSSG特异性结合。[结论]用0.1 mmol/L IPTG于18℃诱导培养20 h,每升菌液可纯化得到约2.3 mg纯度超85%的CtaP蛋白。体外功能研究显示CtaP为谷胱甘肽结合蛋白。
[Objective]To overexpress and purify active CtaP protein and investigate its binding ability to GSH in vitro.[Method]The encoding sequence of CtaP was amplified by PCR and cloned into pWaldo-GFPe plasmid.The recombinant plasmid was named pWaldo-CtaP.pWaldo-CtaP was transformed into E.coli expression strain BL21(DE3).The concentra-tion of inducer IPTG and induction temperature were optimized.The soluble and stable CtaP protein was purified by nickel col-umn and molecular sieve column.[Result]The pWaldo-CtaP plasmid was successfully constructed.The recombinant BL21(DE3)strain was cultured at 18℃with 0.1 mmol/L IPTG for 20 h.The protein expressed well and mainly existed in soluble form.Native PAGE analysis showed that CtaP could bind specifically with GSH or GSSG.[Conclusion]With 0.1 mmol/L IPTG at 18℃for 20 h,about 2.3 mg of CtaP protein with a purity of over 85%could be purified from each liter of bacterial solution.In vitro functional studies have shown that CtaP is a glutathione-binding protein.
作者
张梦
嵇君伟
田奋
张蓓
张艳玲
张玉娟
刘颖
王中山
ZHANG Meng;JI Junwei;TIAN Fen;ZHANG Bei;ZHANF Yanling;ZHANG Yujuan;LIU Ying;WANG Zhongshan(Department of Gynecology,Central Hospital of Xuzhou,Xuzhou 221009;Jiangsu Province Key Laboratory of Anesthesiology,Xuzhou Medical University,Xuzhou 221004;The First Clinical Medical College,Xuzhou Medical University,Xuzhou 221004,China)
出处
《生物技术》
2025年第6期671-676,共6页
Biotechnology
基金
国家自然科学基金项目(81600969)
徐州医科大学附属医院发展基金项目(XYFM202208)。
