表皮生长因子受体蛋白酪氨酸激酶的表达、纯化及活性测定

Expression, purification and bioactivity determination of epidermal growth factor receptor protein tyrosine kinase

目的:采用原核表达体系对表皮生长因子受体(epidermal growth factor receptor,EGFR)受体型酪氨酸激酶(recep-tor tyrosine kinase,RTK)进行体外表达、纯化及活性鉴定。方法:以包含EGFR cDNA的pRK5质粒为模板,PCR选择扩增编码EGFR-RTK的cDNA片段,插入pQE30质粒后转染大肠杆菌M15。IPTG诱导表达融合蛋白,包涵体蛋白经复性后亲和层析法纯化,ELISA方法测定蛋白活性。结果:成功地将933 bp的EGFR-RTK cDNA片段插入载体pQE30中,构建了表达载体pQE30-RTK。经诱导在原核表达系统中以包涵体形式高效表达了相对分子质量为37 000的EGFR-RTK融合蛋白,表达量约占菌体总蛋白的65.2％。复性、纯化后的EGFR-RTK蛋白经ELISA检测,结果发现随着加入蛋白量的增加,酶促反应产物磷酸化酪氨酸的量也逐渐增加,两者具有线性关系。结论:本实验成功地利用原核表达系统表达了具有生物学活性的EGFR-RTK,较传统的昆虫细胞表达系统更为简便、经济。

Objective:To express the epidermal growth factor receptor (EGFR) protein tyrosme kinase(RIR) in E.coli and to detect its bioactivity after purifying the fusion protein. Methods: Using the plasmid pRK5 containing the EGFR cDNA as the template, we selectively amplified the fragment which coding EGFR-RTK. The fragment was then inserted into the prokaryotic expression vector (pQE30) and transformed into competent E. coli cells (M15). Fusion protein expression was induced by IPTG. After renaturation, the protein was purified by affinity chromatography and the bioactivity was examined by ELISA. Results: We successfully inserted EGFR-RTK cDNA fragment of 933 bp into vector pQE30 and constructed the expression vector pQE30-RTK. The fusion protein, with a molecular weight of 37 000, was efficiently expressed as inclusion in the prokaryotic expressive system with the yield of 65. 2% total bacterial protein. After renaturation and purification, the recombinant protein was proven to be bioactive by ELISA. Conclusion: The expression and purification of the bioactive EGFR-RTK in prokaryotic expression system in this study is cheaper and easier than the traditional insect expression system.