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

Protein Expr Purif. 2004 Nov;38(1):37-44. doi: 10.1016/j.pep.2004.07.019.

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.

Publication types

  • Evaluation Study

MeSH terms

  • Amino Acid Substitution*
  • Cell-Free System
  • Dimerization
  • HIV Reverse Transcriptase / chemistry
  • HIV Reverse Transcriptase / isolation & purification*
  • Humans
  • Models, Biological
  • Protein Subunits / chemistry*
  • RNA, Transfer / chemistry
  • RNA, Transfer / isolation & purification
  • Ribonuclease H / chemistry
  • Ribonuclease H / isolation & purification

Substances

  • Protein Subunits
  • RNA, Transfer
  • HIV Reverse Transcriptase
  • Ribonuclease H