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Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase

  1. Author:
    Klarmann, G. J.
    Eisenhauer, B. M.
    Yi, Z.
    Sitaraman, K.
    Chatterjee, D. K.
    Hecht, S. M.
    Le Grice, S. F. J.

  2. Author Address

    NCI, HIV Drug Resistance Program, Frederick, MD 21701 USA. Univ Virginia, Dept Chem, Charlottesville, VA USA. Sci Applicat Int Corp, Frederick, MD USA Le Grice, SFJ, NCI, HIV Drug Resistance Program, Frederick, MD 21701 USA
    1. Year: 2004
    2. Date: NOV
  2. Journal: Protein Expression and Purification
    1. 38
    2. 1
    3. Pages: 37-44
  4. Type of Article: Article
  1. Abstract:

    A highly efficient cell-free translation system has been combined with suppressor tRNA technology to substitute nor-Tyr and 3-fluoro-Tyr in place of Tyr183 at the DNA polymerase active site of p66 of human immunodeficiency virus type I reverse transcriptase (HIV-1 RT). Supplementing the wild-type HIV-1 p51 RT subunit into this translation system permitted reconstitution of the biologically relevant p66/p51 heterodimer harboring Tyr analogs exclusively on the catalytically competent p66 subunit. Addition of an affinity tag at the p66 C-terminus allowed rapid, one-step purification of reconstituted and selectively mutated heterodimer HIV-1 RT via strep-Tactin-agarose affinity chromatography. The purified enzyme was demonstrated to be free of contaminating nucleases, allowing characterization of the DNA polymerase and ribonuclease H activities associated with HIV-1 RT. Preliminary characterization of HIV-1 RTnor-Tyr and HIV-1 RTm-fluoro-Tyr is presented. The success of this strategy will facilitate detailed molecular analysis of structurally and catalytically critical amino acids via their replacement with closely related, unnatural analogs. (C) 2004 Elsevier lnc. All rights reserved

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External Sources

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  2. DOI: 10.1016/j.pep.2004.07.019
  3. View record in Web of Science®
  4. WOS: 000224778800006

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