谭拥军,陈竹林,陈团辉,向勤.人FOXA1蛋白的原核表达、纯化及蛋白互作分析[J].湖南大学学报:自然科学版,2016,43(6):104~108
人FOXA1蛋白的原核表达、纯化及蛋白互作分析
Prokaryotic Expression, Purification, and Protein-interaction Analysis of Human FOXA1 Protein
  
DOI:
中文关键词:  转录因子FOXA1  原核表达  相互作用蛋白
英文关键词:transcrition factor FOXA1  prokaryotic expression  interaction protein
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作者单位
谭拥军,陈竹林,陈团辉,向勤 (湖南大学 生物学院湖南 长沙410082) 
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中文摘要:
      构建FOXA1的原核表达载体,诱导表达并纯化后获得人FOXA1的C端和N端蛋白片段,为寻找FOXA1的互作蛋白提供材料基础.提取人乳腺癌细胞MCF7的总RNA,逆转录成cDNA,设计引物PCR克隆FOXA1的cDNA,再以此为模板扩增FOXA1的C端、N端cDNA片段,分别并克隆进带有GST标签的原核表达载体pGEX-4T-1,构建重组表达质粒.双酶切及测序鉴定正确后,转化至大肠杆菌BL21 Rosetta DE3,经IPTG诱导,Glutathione Sepharose 4B亲和纯化,通过SDS-PAGE电泳及Western blot确定FOXA1蛋白的表达.与MCF7细胞裂解液孵育,进行GST-Pull down试验,检测纯化蛋白与FOXA1已知互作蛋白TLE3的相互作用.成功构建pGEX-4T-1-FOXA1-C和 pGEX-4T-1-FOXA1-N的原核表达载体;在大肠杆菌中诱导目的蛋白表达;经Glutathione Sepharose 4B纯化后,获得了人FOXA1的C端蛋白片段GST-FOXA1-C与N端蛋白片段GST-FOXA1-N;证实GST-FOXA1-C能与FOXA1已知互作蛋白TLE3的相互作用.
英文摘要:
      To identify proteins interacting with transcription factor FOXA1,different prokaryotic expression vectors for human FOXA1 protein were constructed and the proteins of FOXA1 C-terminus and FOXA1 N-terminus were purified. They provided a material foundation for future studies of FOXA1-interacted proteins. Total RNAs were extracted from human breast cancer MCF7 cells and reverse transcribed into cDNAs. The full length cDNA of FOXA1 was amplified by PCR, and the C-terminal and N terminal segments were also amplified respectively. The different FOXA1 cDNA fragments were cloned into a prokaryotic expression vector pGEX-4T-1. The correct vectors were confirmed by double restriction enzyme digestion and DNA sequencing and transformed into E. coli BL21 Rosetta DE3. The FOXA1 proteins were purified with Glutathione Sepharose 4B and analyzed through SDS-PAGE electrophoresis and Western blot. GST Pull-down experiments were performed with lysates of MCF7 cells and FOXA1 proteins to test the interaction between FOXA1 and a known FOXA1-interacted protein TLE3.Prokaryotic expression vectors of pGEX-4T-1-FOXA1-C and pGEX-4T-1-FOXA1-N were constructed successfully. The proteins of FOXA1 C-terminus (GST-FOXA1-C) and FOXA1 N-terminus (GST-FOXA1-N) were purified by Glutathione Sepharose 4B. It was confirmed that the purified GST-FOXA1-C was able to interact with the known FOXA1-interacted protein TLE3 in the Pull-down experiment.
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