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| Kinetic Study of The HIV-1 Integrase 3'-processing Reaction Using a Molecular Beacons Based Assay |
| HE Hong-qiu1,2, LIU Bin1, CHEN Wei-zu1, WANG Cun-xin1 |
1. College of Life Science and Bio-engineering, Beijing University of Technology, Beijing 100124, China;
2. Chongqing Center for Biomedicines and Medical Equipment, Chongqing Academy of Science and Technology, Chongqing 401123,China |
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Abstract HIV-1 integrase (IN) mediates the integration of viral cDNA into the host genome, which is a key process for viral replication. The 3'-processing of viral DNA extremities is the first step in the integration process, and kinetic studies on the 3'-processing reaction will be helpful in mechanism studies of IN and anti-IN drug design. Wild type IN recombinant plasmid was constructed and IN was expressed in E. coli BL21 cell. Subsequently, IN was purified through denaturation and refolding procedure from inclusion bodies. Based on molecular beacons, fluorophore and quencher-labeled DNA substrate was designed and applied in real-time monitoring of 3'-processing reaction as well as subsequent kinetic study of the reaction. As a result, the purified IN was active, and IN prefered Mg2+ as the cationic cofactor in the reaction. The studies on its enzymatic kinetics (Km = 131.79 nmol/L and Kcat = 0.0042 min -1) elucidate that both the molecular beacons based real-time assay and the designed DNA substrate are suited for use in 3'-processing kinetic study as well as other reaction characters studies.
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Received: 29 September 2011
Published: 25 February 2012
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