人纤溶酶原K5突变体Ⅰ的构建及其在大肠杆菌中的表达与纯化

Construction,Expression and Purification of Human Plasminogen Kringle 5 Mutant Ⅰ in E.coli

【目的】 构建人纤溶酶原Kringle 5(K5)的突变体Cys461-Cys540(K5 mut1),在大肠杆菌中表达,亲和层析纯化,为探讨K5抗血管增生活性与Kringle结构域的关系提供基础?【方法】 以K5全长cDNA为模板用PCR的方法扩增人纤溶酶原K5 mut1基因,将其克隆进表达载体pET-22b(+)中,并用限制性核酸内切酶酶切和DNA测序鉴定其连接正确;pET-22b(+)/K5 mut1 转化大肠杆菌BL21(DE3), IPTG诱导表达,表达产物用固化Ni2+-His Bind Resin亲和层析方法纯化,用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western blot方法分析鉴定?【结果】 PCR扩增获得258 bp的人纤溶酶原K5 mut1基因片段,正确插入pET-22 b(+)载体,在大肠杆菌中该基因编码蛋白的表达量占菌体总蛋白13%左右;SDS-PAGE显示其相对分子质量Mr≈14.1×103,Western blot证实该表达蛋白为K5 mut1重组蛋白,经亲和层析后K5 mut1重组蛋白纯度大于90%,获得率约为10 mg/L?【结论】 成功构建人纤溶酶原K5 mut1,实现在大肠杆菌中高效表达,亲和层析纯化获得较高纯度K5 mut1重组蛋白?

Objective The purpose of the present study was to construct, express and purify hu-man plasminogen kringle 5 mutant Ⅰ(K5 mut1).The purified K5 mut1 recombinant protein will be em-ployed to explore the correlation between the anti- angiogenic activity and the kringle domain of K5. Methods The gene of K5 mut1 was amplified from the K5 cDNA by PCR and inserted into the expres-sion vector of pET-22b(+).The recombinant pET-22b(+)/K5 mut1 was transferred into E.coli of BL21(DE3) and identified by restriction enzyme analysis. K5 mut1 was expressed in E.coli induced by IPTG. and Ni2+-His Binding affinity-purified. The recombinant protein was identified by SDS-PAGE and Western blot analysis. Results The acquired mutant gene was 258 bp. The con-struct was expressed in E.coli at high level as soluble protein, accounting for 13% of the total bacteri-al proteins. The relative molecular mass of the protein appeared to be about 14.1×103 by SDS-PAGE. The purity of K5 mut1 reaches up to over 90% after purified by Ni2+-His Bind Resin affini-ty chromatography. The identify of recombinant K5 mut1 was confirmed by Western blot analysis using anti-His tag antibody. An average of 10 mg of purified K5 mut1 was obtained from 1 litre of E.coli cul-ture. Conclusion The construction, expression and purification of K5 mut1 in E.coli was successful.