hVDAC1蛋白在大肠杆菌中的表达纯化及其结构功能的初步表征  

Expression,purification and characterization of hVDAC1 in E.coli

作　　者：吕万胜[1,2] 朱伶俐[1,2] 吴先登 王泽南[1] 郭文洁[3] 高静[3] 华子春[1,2] 郑伟娟[1,2] 

机构地区：[1]南京大学生命科学学院,南京210023 [2]医药生物技术国家重点实验室,南京210023 [3]江苏大学药学院,镇江212013

出　　处：《南京大学学报（自然科学版）》2016年第4期609-618,共10页Journal of Nanjing University（Natural Science）

基　　金：国家自然科学基金(81072712;81373400);江苏省自然科学基金(BK2012710);国家基础科学人才培养基金(J1103512;J1210026)

摘　　要：电压依赖性阴离子通道蛋白(VDAC)是线粒体外膜上最丰富的蛋白,在调节线粒体的物质代谢与能量代谢、以及线粒体介导的细胞凋亡中起重要作用,因而与肿瘤及神经退行性疾病的发生密切相关.VDAC1是三种VDAC变体中表达量最高、分布最广泛的一种.由于VDAC1的高度保守性,因此成为多种疾病治疗包括抗肿瘤治疗的理想靶点.获得足量的VDAC1蛋白是进一步研究其结构功能、筛选与VDAC1相互作用的药物分子的基础.从生物样品中直接分离纯化VDAC1,不仅条件苛刻,而且得率很低.因此利用DNA重组技术在大肠杆菌中重组表达VDAC1是获得足量蛋白、用于后续研究的重要途径.成功构建了C端带有His-tag的hVDAC1的重组蛋白原核表达质粒pET28a/hVDAC1,在大肠杆菌中以包涵体形式表达,并通过盐酸胍变性、在变性条件下通过NTA-Ni亲和层析柱纯化蛋白后进行复性的方法,获得了高纯度的hVDAC1.圆二色谱检测显示该蛋白二级结构以β-折叠为主、符合hVDAC1的二级结构特征.利用流式细胞仪检测结果显示:FITC荧光标记的hVDAC1可以与脂质体膜、细胞膜特异性结合.证明克隆表达纯化的hVDAC1具有特征的膜蛋白结构和功能,为体外筛选与hVDAC1相互作用的各类分子奠定了基础.Voltage-dependent anion channel（VDAC）is the most abundant protein found in the outer membrane of mitochondria,playing crucial roles not only in the regulation of metabolic and energetic functions of mitochondria,butalso in mitochondria-mediated apoptosis,thus involved in the development of certain diseases including cancer and neurodegenerative disorders.Among the three versions of VDAC which have been identified in mammals,VDAC1 is the one with the highest expression level and widely distribution in various tissues.The highly conservative property of VDAC1 makes it an excellent target for therapies of various diseases including anti-cancer intervention.Acquiring enough VDAC1 protein is the first step for further study of its structure and function relationship,as well as the screen of molecules interacting with VDAC1.Isolation of VDAC1 from various tissues encountered apparent contradictions,such as complicated purification processes and low yield.Therefore,expression and purification of recombinant VDAC1 in E.coli is a realistic approach to obtain large amount of protein for further research.In this study,recombinant protein hVDAC1,with C-terminal His-tag,was expressed and purified fromE.coli with high purity.The recombinant protein forms inclusion bodies when expressed in E.coli.After sonication and centrifugation,inclusion bodies were collected and denatured by high concentration guanidine hydrochloride.Purified by NTA-Ni affinity chromatography and renatured by dialysis,hVDAC1 with high purity was obtained.The CD spectrum shows that the purified hVDAC1 adopts a similar folded conformation with highβ-sheet content,consistent with the previous studies.The specific binding of FITC-labeled hVDAC1 to liposome or cell plasma membrane were confirmed by flow cytometry assay.These results show that the hVDAC1 protein we expressed and purified has the characteristic structure and function of membrane protein,and may provide a basis for further study.

关 键 词：电压依赖性阴离子通道蛋白 原核表达 圆二色谱 脂质体 

分 类 号：TP181[自动化与计算机技术—控制理论与控制工程]