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

Authors

  • Kun Silprasit Interdisplinary Graduated Program in Genetic Engineering, Kasetsart University, Bangkok 10900, Thailand.
  • Ratsupa Thammaporn Department of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
  • Supa Hannongbua Interdisplinary Graduated Program in Genetic Engineering, Kasetsart University, Bangkok 10900, Thailand.
  • Kiattawee Choowongkomon Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.

Keywords:

reverse transcriptase, HIV-1, cloning, expression, activity assay

Abstract

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 Km, 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.

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Published

2008-12-31

How to Cite

Kun Silprasit, Ratsupa Thammaporn, Supa Hannongbua, and Kiattawee Choowongkomon. 2008. “Cloning, Expression, Purification, Determining Activity of Recombinant HIV-1 Reverse Transcriptase”. Agriculture and Natural Resources 42 (5). Bangkok, Thailand:231-39. https://li01.tci-thaijo.org/index.php/anres/article/view/244600.

Issue

Vol. 42 No. 5 (2008): January-December

Section

Research Article

License

online 2452-316X print 2468-1458/Copyright © 2022. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/),
production and hosting by Kasetsart University of Research and Development Institute on behalf of Kasetsart University.