|本期目录/Table of Contents|

 Lyu Wansheng,Zhu Lingli,Wu Xiandeng,et al.Expression,purification and characterization of hVDAC1 in E.coli[J].Journal of Nanjing University(Natural Sciences),2016,52(4):609.[doi:10.13232/j.cnki.jnju.2016.04.005]





Expression,purification and characterization of hVDAC1 in E.coli
吕万胜12朱伶俐12吴先登1王泽南1郭文洁3高 静3华子春12*郑伟娟12*
Lyu Wansheng12Zhu Lingli12Wu Xiandeng1Wang Ze’nan1Guo Wenjie3Gao Jing3Hua Zichun12*Zheng Weijuan12*
1.School of Life Science,Nanjing University,Nanjing,210023,China;2.State Key Laboratory of Pharmaceutical Biotechnology,Nanjing,210023,China;3.School of Pharmacology,Jiangsu University,Zhenjiang,212013
voltage­dependent anion channelexpressionCircular Dichroismliposome
Voltage­dependent 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,but also in mitochondria­mediated 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 anti­cancer 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 C­terminal His­tag,was expressed and purified from E.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 NTA­Ni 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 high β­sheet content,consistent with the previous studies.The specific binding of FITC­labeled 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.


[1] Youle R J,Narendra D P.Mechanisms of mitophagy.Nature Reviews Molecular Cell Biology,2011,12(1):9-14.
[2]  Colombini M,Mannella C A.VDAC,the early days.Biochimica et Biophysica Acta,2012,1818(6):1438-1443.
[3]  Shoshan­Barmatz V,De Pinto V,Zweckstetter M,et al.VDAC,a multi­functional mitochondrial protein regulating cell life and death.Molecular Aspects of Medicine,2010,31(3):227-285.
[4]  Shoshan­Barmatz V,Golan M.Mitochondrial VDAC1:Function in cell life and death and a target for cancer therapy.Current Medicinal Chemistry,2012,19(5):714-735.
[5]  Shoshan­Barmatz V,Mizrachi D.VDAC1:from structure to cancer therapy.Frontiers in Oncology,2012,2(164).
[6]  Reddy P H.Is the mitochondrial outermembrane protein VDAC1 therapeutic target for Alzheimer’s disease?Biochimica et Biophysica Acta,2013,1832(1):67-75.
[7]  Benz R.Permeation of hydrophilic solutes through mitochondrial outer membranes:Review on mitochondrial porins.Biochimica et Biophysica Acta,1994,1197(2):167-196.
[8]  Messina A,Reina S,Guarino F,et al.VDAC isoforms in mammals.Biochimica et Biophysica Acta,2012,1818(6):1466-1476.
[9]  Engelhardt H,Meins T,Poynor M,et al.High­level expression,refolding and probing the natural fold of the human voltage­dependent anion channel isoforms Ⅰ and Ⅱ.The Journal of Membrane Biology,2007,216(2-3):93-105.
[10]  Blachly­Dyson E,Zambronicz E B,Yu W H,et al.Cloning and functional expression in yeast of two human isoforms of the outer mitochondrial membrane channel,the voltage­dependent anion channel.The Journal of Biological Chemistry,1993,268(3):1835-1841.
[11]  De Pinto V,Messina A,Lane D J,et al.Voltage­dependent anion­selective channel(VDAC)in the plasma membrane.FEBS Letters,2010,584(9):1793-1799.
[12]  Shoshan­Barmatz V,Hadad N,et al.VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle.FEBS Letters,1996,386(2-3):205-210.
[13]  Shoshan­Barmatz V,Zalk R,Gincel D,et al.Subcellular localization of VDAC in mitochondria and ER in the cerebellum.Biochimica et Biophysica Acta,2004,1657(2-3):105-114.
[14]  Bayrhuber M,Meins T,Habeck M,et al.Structure of the human voltage­dependent anion channel.Proceedings of the National Academy of Sciences of the United States of America,2008,105(40):15370-15375.
[15]  Hiller S,Garces R G,Malia T J,et al.Solution structure of the integral human membrane protein VDAC­1 in detergent micelles.Science,2008,321(5893):1206-1210.
[16]  Ujwal R,Cascio D,Colletier J P,et al.The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating.Proceedings of the National Academy of Sciences of the United States of America,2008,105(46):17742-17747.
[17]  Hiller S,Abramson J,Mannella C,et al.The 3D structures of VDAC represent a native conformation.Trends in Biochemical Sciences,2010,35(9):514-521.
[18]  Geula S,Ben­Hail D,Shoshan­Barmatz V.Structure­based analysis of VDAC1:N­terminus location,translocation,channel gating and association with anti­apoptotic proteins.Biochemical Journal,2012,444(3):475-485.
[19]  Brahimi­Horn M C,Ben­Hail D,Lie M,et al.Expression of a truncated active form of VDAC1 in lung cancer associates with hypoxic cell survival and correlates with progression to chemotherapy resistance.Cancer Research,2012,72(8):2140-2150.
[20]  Bai Z,Ye Y,Liang B,et al.Proteomics­based identification of a group of apoptosis­related proteins and biomarkers in gastric cancer.International Journal of Oncology,2011,38(2):375-383.
[21]  Simamura E,Shimada H,Ishigaki Y,et al.Bioreductive activation of quinone antitumor drugs by mitochondrial voltage­dependent anion channel 1.Anatomical Science International,2008,83(4):261-266.
[22]  Yoo H J,Yun B R,Kwon J H,et al.Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells.Experimental & Molecular Medicine,2009,41(2):102-115.
[23]  Bryson J M,Coy P E,Gottlob K,et al.Increased hexokinase activity,of either ectopic or endogenous origin,protects renal epithelial cells against acute oxidant­induced cell death.The Journal of Biological Chemistry,2002,277(13):11392-11400.
[24]  Pedersen P L,Mathupala S,Rempel A,et al.Mitochondrial bound type II hexokinase:Akey player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.Biochimica et Biophysica Acta,2002,1555(1-3):14-20.
[25]  Koppenol W H,Bounds P L,Dang C V.Otto Warburg’s contributions to current concepts of cancer metabolism.Nature Reviews Cancer,2011,11(5):325-337.
[26]  Shimizu S,Tsujimoto Y.Proapoptotic BH3­only Bcl­2 family members induce cytochrome c release,but not mitochondrial membrane potential loss,and do not directly modulate voltage­dependent anion channel activity.Proceedings of the National Academy of Sciences,2000,97(2):577-582.
[27]  Arbel N,Shoshan­Barmatz V.Voltage­dependent anion channel 1­based peptides interact with Bcl­2 to prevent antiapoptotic activity.The Journal of Biological Chemistry,2010,285(9):6053-6062.
[28]  Shoshan­Barmatz V,Ben­Hail D,Admoni L,et al.The mitochondrial voltage­dependent anion channel 1 in tumor cells.Biochimica et Biophysica Acta,2015,1848(10 Pt B):2547-2575.
[29]  Shoshan­Barmatz V,Ben­Hail D.VDAC,a multi­functional mitochondrial protein as a pharmacological target.Mitochondrion,2012,12(1):24-34.
[30]  Haridas V,Li X,Mizumachi T,et al.Avicins,a novel plant­derived metabolite lowers energy metabolism in tumor cells by targeting the outer mitochondrial membrane.Mitochondrion,2007,7(3):234-240.
[31]  Yang Z,Schumaker L M,Egorin M J,et al.Cisplatin preferentially binds mitochondrial DNA and voltage­dependent anion channel protein in the mitochondrial membrane of head and neck squamous cell carcinoma:Possible role in apoptosis.Clinical Cancer Research,2006,12(19):5817-5825.
[32]  Lai J C,Tan W,Benimetskaya L,et al.A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC.Proceedings of the National Academy of Sciences of the United States of America,2006,103(19):7494-7499.
[33]  Malia T J,Wagner G.NMR structural investigation of the mitochondrial outer membrane protein VDAC and its interaction with antiapoptotic Bcl­xL.Biochemistry,2007,46(2):514-525.






更新日期/Last Update: 2016-07-23