人肿瘤抑制因子folliculin的N端结构原核表达纯化  

Purification of Human Tumor Suppressor Folliculin by Prokaryotic Expression in N-terminal

作　　者：皮业勤 吴哲俊 申一凡 陈婷[1] 陆昌瑞 张云龙[1] Pi Ye-qin;Wu Zhe-jun;Shen Yi-fan;Chen Ting;Lu Chang-rui;Zhang Yun-long(Institute of Biological Science and Technology,Donghua University,Shanghai201620)

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《生物化工》2018年第1期9-12,21,共5页Biological Chemical Engineering

基　　金：国家自然科学基金(№:31300603);国家大学生创新实验项目(№:14T10501);东华大学创新实验计划项目(№:X171051707);东华大学化学化工与生物工程学院创新实验计划项目(№:17T10518)

摘　　要：Folliculin(FLCN)是一种由BHD基因编码的肿瘤抑制因子,BHD基因的缺失或突变会导致BHD综合征,主要临床表现为良性毛囊瘤、肾囊肿和肾肿瘤等,但具体致病机制不明,FLCN的结构与功能有待深入研究。本文基于原核优化的FLCN基因构建N端1-362aa.(N362)的重组蛋白原核表达系统,在0.1mmol/LIPTG和16℃过夜诱导下融合蛋白(His)6-SUMO-N362高效表达,用镍亲和层析和离子交换层析纯化并用ULP1酶切除(His)_6-SUMO标签后,可得到纯度高达80%的N362蛋白,这为后续FLCN的结构和功能分析奠定了良好基础。用生物信息学技术对FLCN氨基酸序列预测,发现N端多无序区域且保守区域100-230aa.有强疏水性,极易形成疏水性片段,推测N端可能作为FLCN形成聚合体或转运结合的区域。Folliculin（FLCN） is a tumor suppressor encoded by BHDgene. The deletion or mutation of BHDgene leads to BHD syndrome. The main clinical manifestations are benign follicular tumors, renal cysts and renal tumors. However, its specific pathogenic mechanism Unknown, the structure and function of FLCN need further study. In this paper, a prokaryotic expression system of N-terminal 1-362 aa.（N362） was constructed based on FLCN gene. The fusion protein（His）_6-SUMO-N362 was efficiently expressed in 0.1 mmol/L IPTG and overnight at 16℃,purification by nickel affinity chromatography and ion exchange chromatography,cutting the His6-SUMO tag with ULP1 and then obtain the N362 protein with a purity up to 80%, which laid a good foundation for the structural and functional analysis of FLCN. We predicted the amino acid sequence of FLCN by bioinformatics technology and found that the N-terminal have abundant disordered region and the conserved region of 100-230 aa. Have strong hydrophobic, which are easily formed hydrophobicfragments, presumably N-terminal may form a polymer or transport as FLCN Combined area.

关 键 词：肿瘤抑制因子 FOLLICULIN 蛋白表达纯化 生物信息学 

分 类 号：Q78[生物学—分子生物学]