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

George J. Klarmann, Brian M. Eisenhauer, Yi Zhang, Kalavathy Sitaraman, Deb K. Chatterjee, Sidney M. Hecht, Stuart F.J. Le Grice

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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 1 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 RT nor-Tyr and HIV-1 RT m-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.

Original languageEnglish (US)
Pages (from-to)37-44
Number of pages8
JournalProtein Expression and Purification
Volume38
Issue number1
DOIs
StatePublished - Nov 2004
Externally publishedYes

Keywords

  • Cell-free transcription/translation
  • Coupled transcription/translation
  • HIV-1
  • Reverse transcriptase
  • Unnatural amino acid

ASJC Scopus subject areas

  • Biotechnology

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