Cloning, Expression, Purification, Determining Activity of Recombinant HIV-1 Reverse Transcriptase

บทคัดย่อ

The spreading of HIV infection is still a serious epidemic disease in Asia, including Thailand, especially by transmission from mother to child and drug resistance HIV. Therefore, discovery of a new drug is the hope for curing the drug resistant strains. Drug discovery needs atomic resolution structure of the target protein, reverse transcriptase. In this paper, we reported the cloning, expression, purification and activity assay of HIV-1 reverse transcriptase in E. coli. The yield of homogeneous recombinant HIV-1 reverse transcriptase enzyme was 2 mg/liter culture. The enzyme activity assay using fluorometric method and PicoGreen dye was convenient and rapid, and K_m, determined by this method was 10.51 ± 2.58 μM dTTP substrate. The fluorometric assay can be further used for high-throughput inhibitor screening application.