八肋游仆虫eRF3 C端的76个氨基酸对于释放因子eRF1a的结合不是必需的

以八肋游仆虫第二类肽链释放因子eRF3基因为模板,用PCR的方法获得eRF3的C端(eRF3C)和C端缺失76个氨基酸的突变体eRF3Ct片段,并构建重组表达质粒pGEX-6p-1-eRF3C和pGEX-6p-1-eRF3Ct,转入大肠杆菌BL21(DE3)中获得了可溶性表达。通过Glutathione Sepharose 4B柱亲和层析纯化,重组蛋白GST-eRF3C和GST-eRF3Ct获得纯化。Western blotting分析表明获得的蛋白为目的蛋白。PreScission酶切割后得到eRF3C和eRF3Ct蛋白。体外pull down分析显示eRF3C和eRF3Ct均能与八肋游仆虫第一类释放因子eRF1a相互作用,这表明八肋游仆虫eRF3 C端的76个氨基酸对于释放因子eRF1a的结合不是必需的。

C-terminal 76 Amino Acids of eRF3 Are Not Required for the Binding of Release Factor eRF1a from Euplotes octocarinatus

Termination of translation in eukaryotes requires two polypeptide chain-release factors, eRF1 and eRF3. eRF1 recog- nizes stop signals, whereas eRF3 is a ribosome-dependent and eRF1-dependent GTPase. Polypeptide release factor eRF3 consists of N-terminal variable region and C-terminal conserved part. C-terminal part of eRF3 is responsible for termination of the translation. In the present study, the C-terminal of Euplotes octocarinatus eRF3 (eRF3C) and truncate eRF3C lacking 76 amino acids in C-terminal (eRF3Ct) were expressed in Escherichia coli. The recombinant GST-eRF3C and GST-eRF3Ct polypeptides were puri- fied by affinity chromatography using glutathione Sepharose 4B column. After enzymatic cleavage of GST tail, the eRF3C and eRF3Ct protein were obtained. Pull-down analysis showed that the recombinant GST-eRF3C and GST-eRF3Ct polypeptides inter- acted with E. octocarinatus polypeptide chain release factor eRF1a. This result suggested that the C-terminal of eRF3 having 76 amino acids were not required for the binding of eRF1a in Euplotes octocarinatus.