三角褐指藻F-box like蛋白基因的原核表达及蛋白纯化  

Prokaryotic Expression and Protein Purification of F-box Like Protein Gene from Phaeodactylum tricornutum

作　　者：黄静依 孙双 张婷 李锐[1] 兰利琼[1] 卿人韦[1] Huang Jingyi;Sun Shuang;Zhang Ting;Li Rui;Lan Liqiong;Qing Renwei(Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education,College of Life Sciences,Sichuan University,Chengdu,610065)

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610065

出　　处：《分子植物育种》2021年第12期3829-3836,共8页Molecular Plant Breeding

基　　金：国家自然科学基金项目(40976092);四川省科技厅项目(2014JY0171)共同资助。

摘　　要：F-box蛋白是SCF复合体的重要组成元件之一,能通过泛素蛋白酶体途径(ubiquitin-proteasome system,UPS)参与调控胞内蛋白降解、受体识别、信号传导等生物学过程。为了探究一个F-box like蛋白在三角褐指藻细胞周期调控过程中的作用机制,本研究从三角褐指藻中克隆得到一个F-box like蛋白基因,构建原核表达载体Tph-pET28a-F-box,将其转入大肠杆菌BL21(DE3)菌株中诱导表达,并对其诱导表达条件进行了优化,最后使用镍螯合亲和层析柱(Ni-NTA Agarose)对重组蛋白进行纯化并进行了生物信息学分析。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)检测。结果显示,导入大肠杆菌BL21(DE3)的三角褐指藻F-box like蛋白基因能够被诱导表达,重组可溶性蛋白最佳诱导条件为16℃时,以浓度为0.1 mmol/L的异丙基β-D-硫代半乳糖苷(IPTG)诱导12 h,纯化后的蛋白通过Western blot鉴定,显示在相对分子质量约为72.75 kD处出现一条与预期大小相符的蛋白印迹条带。生物信息学分析结果表明,该蛋白具有F-box蛋白的典型特征,位于膜外且不含有跨膜结构域,为亲水性蛋白,α-螺旋和无规卷曲为三角褐指藻F-box蛋白二级结构的主要组成元件。本研究为进一步研究三角褐指藻F-box like蛋白的作用机制奠定了基础。F-box protein is one of the important components of the SCF complex.It can participate in the regulation of intracellular protein degradation,receptor recognition,and signal transduction through the ubiquitin-proteasome system(UPS).In order to explore the mechanism of action of an F-box like protein gene in the cell cycle regulation of Phaeodactylum tricornutum,this study cloned an F-box like protein gene from P.tricornutum,then the prokaryotic expression vector pET-28 a-F-box was constructed by gene recombination technology,which was transformed into E.coli BL21(DE3)strain for expression as well as optimized expression conditions.Finally,the recombinant protein was purified using Ni-NTA Agarose and then bioinformatics analysis was performed.Detection by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)showed that the F-box like protein gene of P.tricornutum introduced into E.coli BL21(DE3)can be induced to express.The optimal induction conditions for the recombinant protein were induced by isopropylβ-D-thiogalactoside(IPTG)at a concentration of 0.1 mmol/L for 12 h at 16℃,the purified protein was identified by Western blot and showed a Western blotting band at a relative molecular mass of 72.75 kD,which was consistent with the expected size.The results of bioinformatics analysis show that the protein has the typical characteristics of F-box protein,is located outside the membrane and does not contain a transmembrane domain,is a hydrophilic protein,α-helix and random coils are the main components of the secondary structure.This study lays the foundation for further studying the mechanism of F-box like protein of P.tricornutum.

关 键 词：三角褐指藻(Phaeodactylum tricornutum) F-box like蛋白 原核表达 蛋白纯化 蛋白质印迹法 

分 类 号：Q943.2[生物学—植物学]