药学学报, 2014, 49(8): 1200-1207
引用本文:
张大为, 何红秋, 郭顺星. LEDGF/p75蛋白的可溶性表达、纯化及功能研究[J]. 药学学报, 2014, 49(8): 1200-1207.
ZHANG Da-wei, HE Hong-qiu, GUO Shun-xing. Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein[J]. Acta Pharmaceutica Sinica, 2014, 49(8): 1200-1207.

LEDGF/p75蛋白的可溶性表达、纯化及功能研究
张大为1, 何红秋2, 郭顺星1
1. 中国医学科学院、北京协和医学院药用植物研究所, 北京 100193;
2. 重庆市科学技术学院重庆生物医药与器械研究中心, 重庆 401123
摘要:
HIV-1整合酶(integrase,IN) 是病毒复制过程中的一个关键酶,其与宿主晶状体上皮源性生长因子p75(lens epithelium-derived growth factor p75,LEDGF/p75) 的蛋白-蛋白相互作用是筛选抗病毒药物的一个重要靶点。为开展以IN-LEDGF/p75相互作用为靶点的抑制剂研究,本研究构建了LEDGF/p75蛋白重组质粒,在原核细胞中进行了可溶性表达和功能研究。根据大肠杆菌密码子偏爱性,全合成高利用率密码子的LEDGF/p75基因序列,并克隆到表达载体pGEX-4T-1中构建重组质粒。在大肠杆菌中优化表达LEDGF/p75蛋白,经SDS-PAGE鉴定和亲和色谱纯化蛋白,采用酶联免疫吸附实验方法(ELISA) 测定了LEDGF/p75蛋白的生物学活性。结果显示,构建的重组质粒获得高效稳定的可溶性表达,ELISA证实体外表达的LEDGF/p75蛋白能够与HIV-1 IN相互作用,并促进IN链转移反应的完成。本研究为建立以LEDGF/p75-IN相互作用为靶点的抗HIV药物筛选平台打下了基础。
关键词:    LEDGF/p75      基因表达      HIV-1整合酶      ELISA     
Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein
ZHANG Da-wei1, HE Hong-qiu2, GUO Shun-xing1
1. Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, China;
2. Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123, China
Abstract:
HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then trans-formed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2++ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to en-hance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.
Key words:    LEDGF/p75    gene expression    HIV-1 integrase    ELISA   
收稿日期: 2014-02-11
基金项目: 国家自然科学基金资助项目(31170016,81202438).
通讯作者: 郭顺星 Tel/Fax:86-10-62829619,E-mail:sxguo1986@163.com
Email: sxguo1986@163.com
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参考文献:
[1] Liano M, Morrison J, Poeschla EM. Virological and cellular roles of the transcriptional coactivator LEDGF/p75 [J]. Curr Top Microbiol Immunol, 2009, 339: 125-146.
[2] Dietz F, Franken S, Yoshida K, et al. The family of hepatomaderived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies [J]. Biochem J, 2002, 366: 491-500.
[3] Al-Mawsawi LQ, Neamati N. Blocking interactions between HIV-1 integrase and cellular cofactors: an emerging antiretroviral strategy [J]. Trends Pharmacol Sci, 2007, 28: 526-535.
[4] Chen WM, Liu XY. LEDGF/p75: a novel target for anti-HIV therapy and advances in the study of its related inhibitors [J]. Acta Pharm Sin (药学学报), 2009, 44: 953-960.
[5] Maertens G, Cherepanov P, Pluymers W, et al. LEDGF/p75 is essential for nuclear and chromosomal targeting of HIV-1 integrase in human cells [J]. J Biol Chem, 2003, 278: 33528-33539.
[6] Busschots K, Voet A, De Maeyer M, et al. Identification of the LEDGF/p75 binding site in HIV-1 integrase [J]. J Mol Biol, 2007, 365: 1480-1492.
[7] Ciuffi A, Llano M, Poeschla E, et al. A role for LEDGF/p75 in targeting HIV DNA integration [J]. Nat Med, 2005, 11: 1287-1289.
[8] De Rijck J, Vandekerckhove L, Gijsbers R, et al. Overex-pression of the lens epithelium-derived growth factor/p75 integrase binding domain inhibits human immunodeficiency virus replication [J]. J Virol, 2006, 80: 11498-11509.
[9] Emiliani S, Mousnier A, Busschots K, et al. Integrase mutants defective for interaction with LEDGF/p75 are im-paired in chromosome tethering and HIV-1 replication [J]. J Biol Chem, 2005, 280: 25517-25523.
[10] Di Santo R. Inhibiting the HIV integration process: past, present, and the future [J]. J Med Chem, 2014, 56: 8588-8598.
[11] Llano M, Saenz, DT, Meehan A, et al. An essential role for LEDGF/p75 in HIV integration [J]. Science, 2006, 314: 461-464.
[12] Schrijvers R., De Rijck J, Demeulemeester J, et al. LEDGF/p75-independent HIV-1 replication demonstrates a role for HRP-2 and remains sensitive to inhibition by LEDGINs [J]. PLoS Pathog, 2012, 8, e1002558.
[13] Shun MC, Daigle JE, Vandegraaff N, et al. Wild-type levels of human immunodeficiency virus type 1 infectivity in the absence of cellular emerin protein [J]. J Virol, 2007, 81: 166-172.
[14] Christ F, Shaw S, Demeulemeester J, et al. Small molecule inhibitors of the LEDGF/p75 binding site of integrase (LEDGINs) block HIV replication and modulate integrase multimerization [J]. Antimicrob Agents Chemother, 2012, 56: 4365-4374
[15] Christ F, Voet A, Marchand A, et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication [J]. Nat Chem Biol, 2010, 6: 442-448.
[16] De Luca L, Barreca ML, Ferro S, et al. Pharmacophore-based discovery of small molecule inhibitors of protein-protein interactions between HIV-1 integrase and cellular cofactor LEDGF/p75 [J]. ChemMedChem, 2009, 4: 1311-1316.
[17] Du L, Zhao Y, Chen J, et al. D77, one benzoic acid derivative, functions as a novel anti-HIV-1 inhibitor targeting the interaction between integrase and cellular LEDGF/p75 [J]. Biochem Biophys Res Commun, 2008, 375: 139-144.
[18] Kessl JJ, Jena N, Koh Y, et al. A multimode, cooperative mechanism of action of allosteric HIV-1 integrase inhibitors [J]. J Biol Chem, 2012, 287: 16801-16811.
[19] Malet I, Delelis O, Valantin MA, et al. Mutations associated with failure of raltegravir treatment affect integrase sensitivity to the inhibitor in vitro [J]. Antimicrob Agents Chemother, 2008, 52: 1351-1358.
[20] Baldanti F, Paolucci S, Gulminetti R, et al. Early emergence of raltegravir resistance mutations in patients receiving HAART salvage regimens [J]. J Med Virol, 2010, 82: 116-122.
[21] He HQ, Ma XH, Liu B, et al. A novel high-throughput format assay for HIV-1 integrase strand transfer reaction using magnetic beads [J]. Acta Pharmacol Sin, 2008, 29: 397-404.
[22] Hou Y, McGuinness DE, Prongay AJ, et al. Screening for antiviral inhibitors of the HIV integrase-LEDGF/p75 interaction using the AlphaScreen luminescent proximity assay [J]. J Biomol Screen, 2008, 13: 406-414.
[23] Cherepanov P, Maertens G, Proost P, et al. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells [J]. J Biol Chem, 2003, 278: 372-381.
[24] Busschots K, Vercammen J, Emiliani S, et al. The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding [J]. J Biol Chem, 2005, 280: 17841-17847.
[25] Van Maele B, Busschots K, Vandekerckhove L, et al. Cellular co-factors of HIV-1 integration [J]. Trends Biochem Sci, 2006, 31: 98-105.
[26] Christ F, Debyser Z. The LEDGF/p75 integrase interaction, a novel target for anti-HIV therapy [J]. Virology, 2013, 435: 102-109.
[27] Yang JL, Gao LL, Zhu P, et al. Codon optimization and eukaryotic expression analysis of the analgesic peptide gene BmK AngM1 from Buthus martensii Karsch [J]. Acta Pharm Sin (药学学报), 2012, 47: 1389-1393.
[28] Yang JL, He HX, Zhu HX, et al. Optimization on the production of analgesic peptide from Buthus martensii Karsch in Pichia pastoris [J]. Acta Pharm Sin (药学学报), 2009, 44: 91-94.
[29] Schmidt FR. Recombinant expression systems in the pharmaceutical industry [J]. Appl Microbiol Biotechnol, 2004, 65: 363-372.
[30] Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli [J]. Microbiol Rev, 1996, 60: 512-538.
[31] Mellor J, Dobson MJ, Roberts NA. Homologous gene expression in Saccharomyces cerevisiae [J]. Gene, 1985, 33: 215-226.
[32] Sandhu KS, Pandey S, Maiti S, et al. GASCO: genetic algorithm simulation for codon optimization [J]. In Silico Biol, 2008, 8: 187-192.
[33] Gao W, Rzewski A, Sun H, et al. UpGene: application of a web-based DNA codon optimization algorithm [J]. Biotechnol Prog, 2004, 20: 443-448.
[34] Cai Y, Sun J, Wang J, et al. QPSOBT: one codon usage optimization software for protein heterologous expression [J]. Bioinformatics, 2010, 2: 25–29.
[35] Gustafsson C, Govindarajan S, Minshull J. Codon bias and heterologous protein expression [J]. Trends Biotechnol, 2004, 22: 346-353.
[36] Craigie R. The molecular biology of HIV integrase [J]. Future Virol, 2012, 7: 679-686.
[37] Cherepanov P. LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro [J]. Nucleic Acids Res, 2007, 35: 113-124.
[38] Wang H, Shun CM, Li X, et al. Efficient transduction of LEDGF/p75 mutant cells by complementary gain-of-function HIV-1 integrase mutant viruses [J]. Mol Ther -Methods Clin Dev, 2014, doi:10.1038/mtm. 2013.2.
[39] Busschots K, Vercammen J, Emiliani S, et al. The interaction of LEDGF/p75 with integrase is lentivirus-specific and promotes DNA binding [J]. J Biol Chem, 2005, 280: 17841-17847.
[40] Raghavendra NK, Engelman A. LEDGF/p75 interferes with the formation of synaptic nucleoprotein complexes that catalyze full-site HIV-1 DNA integration in vitro: implications for the mechanism of viral cDNA integration [J]. Virology, 2007, 360: 1-5.