人体中可溶性VEGFR-2酪氨酸激酶在大肠杆菌中的表达、纯化及活性测定

Purification and Identification of the Soluble VEGFR-2 Kinases Expressed in Escherichia coli

针对人体血管内皮生长因子受体2(Vascular endothelial growth factor receptor 2,VEG-FR-2)受体型酪氨酸激酶(Receptor tyrosine kinase,RTK)进行体外表达,并对表达和纯化条件进行优化。构建表达VEGFR-2酪氨酸激酶重组大肠杆菌,通过研究不同诱导温度、IPTG浓度、诱导时间和超声条件等对蛋白表达的影响,采用Ni-NTA亲和层析法、Western Blot法ELISA方法对重组蛋白进行纯化、鉴定和活性测定。成功地将1 000 bp左右的VEGFR-2酪氨酸激酶DNA片段插入载体pQE30中,在优化条件下重组蛋白较好地可溶性表达。SDS-PAGE表明重组蛋白相对分子质量约为36 000,与预期一致,Western Blot分析表明它能与抗Flk-1和抗His抗体特异反应,ELISA检测结果表明重组蛋白具有利用ATP催化底物磷酸化的激酶活性。通过重组DNA技术使人体VEGFR-2酪氨酸激酶在大肠杆菌得到可溶性表达,纯化重组蛋白具有较高的活性。

To improve the soluble expression of VEGFR-2 tyrosine kinase in Escherichia coli for further study on its biological activity.Different concentrations of IPTG,inducing times and temperatures are used to optimize the expression condition for the soluble recombinant VEGFR-2 tyrosine kinase in Escherichia coli.The recombinant protein is purified by affinity chromatography.Western Blot was used to identify this protein.The recombinant plasmid pQE30-TK encoding VEGFR-2 tyrosine kinase was constructed and soluble VEGFR-2 tyrosine kinase was expressed.SDS-PAGE analysis showed that the molecular mass of VEGFR-2 tyrosine kinase was about 36 000 as expected and Western blot analysis indicated that both antibodies for anti-Flk-1 and anti-His could react against the purified protein.Conclusion： The soluble human VEGFR-2 tyrosine kinase was expresses in Escherichia coli by the recombinant technique.The purified protein showed biological activity.