Bioactive And Kinetic Analysis Of HIV-1 Integrase

Integrase is an enzyme encoded by the 3' end of the human immunodeficiency virus type 1(HIV-1)/pol gene, which is proteolytic cleavage product of a Pr160^gagpol fusion precursor protein. Integrase catalyzes the integration of a DNA copy of the viral genome into the host cellular chromosome. It is essential for replication of the virus and is required for stable and productive infection. The integrase has no counterpart in the host cells and is therefore, a suitable target for drug therapeutic intervention. Retro viral DNA integration involves a defined set of DNA cutting and joining reactions. Integrase possesses four different activities: DNA-binding, end-processing, transfer of donor DNA to acceptor DNA (integration) and disintegration have been demonstrated . After binding to the double-stranded blunt-ended viral DNA integrase cleaves the two 3'terminal bases and then joins the ends to acceptor DNA molecule, The strand transfer reaction is reversible, when presented with a substrate similar to 3'-end joining product, the reaction can reverse itself to produce the separate viral and target DNA components. Crystal structure analysis defines the three main domains within the HIV-1 integrase protein. These are the N-terminal domain including a zinc finger like region, the central region containing the catalytic site, and the C-terminal region with a DNA-binding domain. The most common assay uses a radiolabeled oligonucleotide substrate which, after incubation with integrase, is loaded on a denaturing gel and electrophoresed, followed by autoradiography forrevealing the reaction products. This method is time-consuming, inconvenient, andtedious. We aimed to develop a simple, effective and safe method for high throughputscreening of inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase.[Methods]The sequence encoding the full length of HIV-I integrase has been highly effectivelyexpressed in E.coli BL21 (DE3) using recombinant expression plasmidPT7-His-TX-WT-IN. The expression product mainly existed in inclusion bodies. Afterwash and recovery, the inclusion bodies were purified in one step by affinitychromatography on a Ni2+-charged chromatography resin, thus, highly pure andsoluble integrase was acquired. A in vitro integrase assay based to Biotin-AvidinEILSA was adopted to evaluate the bioactivity and kinetic parameters of integrase aswell as the inhibition of luffin-a, one of the ribosome inactivating proteins type 1 (RIPI),on the integrase.[Results]After expression, recovery and purification, we obtained the integrase protein with itspurity more than 85% analysized by SDS-PAGE. In the integrase assay, the integraseexhibited favorable biologic activity. By measurement and calculation, we aquiredthese results: ? the specific activity of the purified integrase was 54.92 units/mg ofprotein. ?The apparent first-order reaction rate constant was 0.0018 X 10"6L/min.molin the integration reaction. (3) IC50 (concentration causing 50% inhibition ofintegrase) of luffin was 0.63 ± 0.026 uM.[Conclusions]Recombinant HIV-1 integrase is characteristic of potent activity of 3' cleavage activityfor viral DNA and strand transference into host DNA. The results have demonstratedthe non-radioactive assay is feasible for high throughput screening and kineticevaluating of inhibitors of integrase.