稀有密码子影响人RNaseH-Apaf1融合蛋白原核表达

通过限制性内切酶克隆法构建两个pET-28a-TAT-RNaseH-Apaf1重组质粒,其中一个质粒经过稀有密码子的优化。将两个重组质粒分别转化原核表达宿主菌BL21(DE3)和Transetta(DE3),通过SDS-PAGE观察相应融合蛋白诱导表达的情况,并用His标签抗体和Apaf1抗体通过Western Blotting鉴定诱导蛋白。菌液PCR和测序证明经稀有密码子优化和未经稀有密码子优化的人源RNaseH-Apaf1序列成功重组到pET-28a-TAT载体中;经稀有密码子优化的重组质粒成功地在大肠杆菌宿主菌中表达RNaseH-Apaf1融合蛋白,并用Western Blotting检测到该融合蛋白。结果表明,稀有密码子可影响人RNaseH-Apaf1融合蛋白的原核表达。

Effect of Rare Codon on Prokaryotic Expression of Human RNaseH-Apaf1 Fusion Protein

Two pET-28a-TAT-RNaseH-Apaf1 plasmids were constructed by restriction site-dependent cloning,one of which was codon-optimized.These recombinants were transformed into two E.coli hosts,BL21(DE3)and Transetta(DE3)respectively,and their associated proteins were expressed.The RNaseH-Apaf1 infusion protein was examined by SDS-PAGE and detected by western blotting,using His-tag antibody and Apaf1 antibody.Two recombinants,the old pET-28a-TAT-RNaseH-Apaf1 plasmid and the new pET-28a-TAT-RNaseH-Apaf1 plasmid,were identified effectively by bacterial liquid PCR and DNA sequencing analysis.Human RNaseH-Apaf1 fusion protein was expressed successfully because of the codon optimization and confirmed by western blotting.Rare codon affects the prokaryotic expression of human RNaseH-Apaf1 fusion protein.