端粒酶反转录功能区基因的表达和纯化

Expression and Purification of Telomerase Resever Transcriptase Motifs in E.coli

通过PCR技术扩增出hTRT的目的片段,克隆至表达载体pET28 b中并转化至BL21(DE3),经IPTG诱导后在52kDa处发现有外源基因的表达,密度扫描显示表达蛋白含量为20%。目标蛋白以包含体形式存在,含8mol/L尿素和10mmol/LDTT的裂解缓冲液溶解的包含体采用金属螯合层析有效分离出目标蛋白。免疫印迹实验表明:诱导出的融合蛋白是端粒酶反转录功能区基因所编码的蛋白质。

our project is designed to clone a 1.3kb gene fragment of telomerase catalytic subunit gene which contains seven reverse transcriptase motifs and specific region with conserved sequence termed 'T motif'.The gene fragment was amplified by PCR and was inserted into expression vector pET28b.The recombinant plasmid was induced by IPTG for 4h and a 52KD recombinant protein was produced.Amount of hTRT recombinant protein expression was 20% of total bacterial protein in the form of inclusion.Inclusion was dissolved in 8mol/L urea and 10mmol/L DTT and carried out affinity purification under denaturing condition.The purified hTRT recombinant protein was conformed by Westernblot successfully.