游仆虫第一类肽链释放因子在大肠杆菌中的表达、纯化和鉴定

Expression, Purification and Identification of eRF1a of Euplotes octocarinatus

为对肽链释放因子结构与功能进行研究 ,进而探讨纤毛虫这类生物中遗传密码表达特殊性的机理 ,利用PCR技术和基因重组技术构建了游仆虫第 1类肽链释放因子eRF1a及C端带 6个组氨酸的eRF1a(His) 6的两个重组表达质粒pBV2 2 1 eRF1a和pBV2 2 1 eRF1a(His) 6.在大肠杆菌DH5α中 ,通过 4 2℃高温诱导 3h ,eRF1a和eRF1a(His) 6获得了可溶性表达 .eRF1a(His) 6的表达水平达到可溶性细菌总蛋白约 8% ,经Ni NTA亲和层析和HitrapQ离子交换层析 ,得到纯度较好的eRF1a(His) 6.

To study the structure and function of polypeptide release factors and understand the mechanism of the genetic deviation of ciliates from other eukaryotes by their reassignment of one or two stop codons to encode amino acids, two expression plasmids pBV221 eRF1a and pBV221 eRF1a(His) 6 were constructed respectively by means of PCR and recombinant DNA techniques. Non fusion and fusion expression of eRF1a in E.coli DH5α were performed by induction at high temperature of 42℃. The expression level of eRF1a(His) 6 was 8% of the total bacterial proteins. The eRF1a(His) 6 was purified by Ni NTA affinity chromatography and ion exchange chromatography. The expression products were also checked by Western blotting assay.