小鼠受体酪氨酸激酶EphB2 FN结构域在大肠杆菌中的表达与纯化

Expression and purification of mouse receptor tyrosine kinase EphB2 FN domain in E.coli

目的：将小鼠受体酪氨酸激酶EphB2 FN结构域在大肠杆菌细胞中进行表达与纯化，以便于研究与FN结构域相互作用的蛋白质。方法以重组质粒GV287－EphB2作为模板，经PCR扩增，得到FN结构域的cDNA片段，并将其克隆至pET－28a载体中。将重组质粒pET－28a－FN转化至大肠杆菌BL21（ DE3）感受态细胞，经IPTG诱导后，利用SDS－PAGE检测FN－His6融合蛋白的表达。最后采用Ni－NTA亲和层析法对该蛋白进行纯化。结果本研究成功地构建了FN结构域的原核表达载体。经鉴定，FN－His6融合蛋白能够在大肠杆菌中表达。纯化后，得到少量可溶性FN－His6融合蛋白。结论小鼠受体酪氨酸激酶EphB2 FN结构域可以在大肠杆菌中表达并纯化，为研究与FN结构域相互作用的蛋白奠定了实验基础。

Objective To express and purify the FN domain of mouse receptor tyrosine kinase EphB2 in E.coli, in order to research the proteins that interacted with this domain.Methods Using the recombinant plasmid GV287 -EphB2 as a template, the cDNA fragment of EphB2 FN domain was amplified by PCR and cloned into pET-28a plas-mid.The resultant recombinant plasmid pET-28a-FN was then transformed into E.coli BL21 （DE3） competent cells. After induction by IPTG, the expression of FN-His6 fusion protein was detected by SDS-PAGE.Finally, Ni-NTA af-finity chromatography was applied to purify FN-His6 .Results The prokaryotic expression vector expressing FN domain was successfully constructed.The expression of FN-His6 fusion protein was determined in E.coli.After purification, a small amount of FN-His6 fusion protein was obtained.Conclusion The FN domain of EphB2 can be expressed and pu-rified in E.coli, which provides experimental basis for researching on the proteins that interact with the domain.