Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase.
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.
HIV Drug Resistance Program, National Cancer Institute-Frederick, Frederick, MD, USA.
George J
GJ
Klarmann
Brian M
BM
Eisenhauer
Yi
Y
Zhang
Kalavathy
K
Sitaraman
Deb K
DK
Chatterjee
Sidney M
SM
Hecht
Stuart F J
SF
Le Grice
D019943:Amino Acid Substitution; D002474:Cell-Free System; D019281:Dimerization; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D008954:Models, Biological; D021122:Protein Subunits / Q000737:chemistry*; D012343:RNA, Transfer / Q000737:chemistry / Q000302:isolation & purification; D016914:Ribonuclease H / Q000737:chemistry / Q000302:isolation & purification
D021122:Protein Subunits; D012343:RNA, Transfer; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H