title: [Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein].
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 transformed 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 enhance 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.
authors.forename: Da-Wei
authors.initials: DW
authors.lastname: Zhang
authors.forename: Hong-Qiu
authors.initials: HQ
authors.lastname: He
authors.forename: Shun-Xing
authors.initials: SX
authors.lastname: Guo
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D019427:HIV Integrase / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis*; D011485:Protein Binding; D014779:Virus Replication
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; C119380:lens epithelium-derived growth factor; D019427:HIV Integrase; C514801:p31 integrase protein, Human immunodeficiency virus 1