中国林蛙核糖核酸酶Rdrlec新基因的原核可溶表达

Prokaryotic Soluble Expression of a Novel Ribonuclease Gene Rdrlec from Rana dybowskii

来源于蛙属的核糖核酸酶由于具有显著的抗肿瘤活性而备受关注,Rdrlec是从中国林蛙基因组中克隆得到的核糖核酸酶新基因。获得大量高纯度野生型重组蛋白是研究其功能的基础。按照大肠杆菌偏好的密码子人工合成Rdrlec基因,通过EcoR I和Hind III位点插入到表达载体pET-32a(+)中构建pET32-Rdrlec重组表达质粒,转化到Escherichia coli BL21(DE3)中,0.4 mmol/L IPTG 30℃诱导6 h后,融合蛋白主要以可溶形式表达,经过Ni-NTA亲和纯化和Sephadex G75层析纯化,得到电泳纯融合蛋白。肠激酶切割后得到Rdrlec野生型重组蛋白,具有降解RNA的酶活性,证明分子的空间结构已经正确形成。Rdrlec野生型重组蛋白表达成功,为后续蛋白结构与功能的研究以及进一步的开发应用提供了原料。

Much attention has been paid to ribonucleases from amphibian Rana species for their significant anti-tumor activity.Rdrlec is a novel RNase gene form Rana dybowskii and its biological function has not been identified.Getting a great deal of high purity wild type recombinant protein is the basis for its function study.Rdrlec gene was adjusted according to Escherichia coli codon bias without changing its amino acids.The synthetic gene was inserted to the pET-32a（＋） expression vector through the EcoR I and Hind III site,and the resulting recombination expression plasmid was named pET32-Rdrlec and transferred into Escherichia coli BL21（DE3） strains.After induced with 0.4 mmol/L IPTG at 30 ℃ for 6 h,the fusion protein was found expressed mainly in soluble form.The cell lysate was loading to Ni-NTA affinity chromatography and Sephadex G75 chromatography,and the fusion protein showed a single band on SDS-PAGE.The wild type recombinant Rdrlec protein was released and purified after enterokinase digesting,and it showed enzymatic activity to degrade RNA into nucleotides,which shows that this molecule has formed the correct spatial structure.The successful expression of wild type recombinant Rdrlec protein has providing the raw material for the subsequent structure,function and application study.