Cloning, Expression, Purification, Activity Assay of Recombinant HIV-1 Reverse Transcriptase in Escherichia coli การทาํใหบรสิุทธิ์และทดสอบ Activity ของโปรตีน HIV-1 Reverse Transcriptase ที่ผลิตโดยเชื้อ Escherichia coli
نویسندگان
چکیده
The spreading of HIV infection still is a serious epidemic disease in the Asia, including Thailand, especially by transmission from mother to child and resistant HIV strains causing the tolerant drug treatment. Therefore, discovery of a new drug is the hope for curing resistant strains which need atomic resolution structure of target protein, reverse transcriptase. In this paper, we reported the cloning, expression, purification and activity assay of HIV-1 reverse transcriptase in E. coli. 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 Km, determined by this method was 15 uM dTTP substrate. The fluorometric assay can be further used for high-throughput inhibitor screening application.
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