hβ-arrestin1重组蛋白不同亚型筛选及互作鉴定

Identification of hβ-arrestin1 Isoform A,B and Recombinant Proteins Interaction with p80

目的构建GST/β-arrestin1融合蛋白表达载体,筛选其不同亚型重组子,并诱导表达、纯化蛋白,初步进行蛋白亚型互作比较,为进一步功能研究提供实验基础。方法提取人源细胞的mRNA,反转录为cDNA。用PCR法扩增hβ-arrestin1全长编码基因,通过SalI和NotI酶切位点将hβ-arrestin1全长定向插入pGEX-5X-1中,构建原核表达载体pGEX-5X-1-β-arrestin1,并通过BglⅡ单酶切筛选A、B亚型,然后将重组质粒转入E.coli BL21中,异丙基硫代β-D半乳糖苷诱导表达,用谷胱甘肽-琼脂糖珠亲和纯化表达的GST/β-arrestin1A、B,SDS-PAGE鉴定,互作实验比较亚型的结合能力。结果酶切及测序结果证明,成功构建了β-arrestin1A、B原核表达载体,并通过SDS-PAGE证实:经IPTG诱导表达及亲和纯化,从转化重组质粒的BL21菌株中获得了融合蛋白GST/β-arrestin1A、B亚型,通过GST Pulldown初步比较了重组β-arrestin1A、B蛋白亚型的结合能力。结论成功构建pGEX-5X-1-β-arrestin1A、B原核表达载体并确定了融合蛋白的诱导表达及纯化方法,且互作实验显示β-arrestin1A有较强的结合能力,为进一步研究β-arrestin1的生物学功能奠定了基础。

Objective To construct GST/β-arrestinl expression vectors, select different recombinant isoforms,and harvest purified fusion protein from E.coh＇. Methods Total RNA was extracted from human epithelial cells. The hβ-arrestinl coding sequence was amplified by polymerase chain reaction（PCR）. The full length of hβ-arrestinl was directed into the pGEX-5X-1 ,to construct prokaryotic expression vector. Further, to enzymatically select recombinant subtype A, B, which were transformed into E.coli BL21 ,induced by IPTG and purified with glutathione beads. The purified products were analyzed by SDS-PAGE. Results Recombinant of pGEX-5X-1-β-arrestinlA,B were analyzed by restriction enzyme digestion and sequenced. GST/β-arrestin1 A, B induced by IPTG were purified from strain BL21. GST/β-arrestin1 A, B were confirmed by SDS-PAGE. GST Pulldown was employed to comfirm binding capacity of GST/β-arrestinl A, B. Conclusion Recombinant GST/β-arrestinl A,B expression vectors were successfully constructed,the specific recombinant isoforms induced were detected,and harvested with high purity for the further study of the biological function of β-arrestin1.