TY - JOUR
T1 - Site- and subunit-specific incorporation of unnatural amino acids into HIV-1 reverse transcriptase
AU - Klarmann, George J.
AU - Eisenhauer, Brian M.
AU - Zhang, Yi
AU - Sitaraman, Kalavathy
AU - Chatterjee, Deb K.
AU - Hecht, Sidney M.
AU - Le Grice, Stuart F.J.
PY - 2004/11
Y1 - 2004/11
N2 - 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.
AB - 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.
KW - Cell-free transcription/translation
KW - Coupled transcription/translation
KW - HIV-1
KW - Reverse transcriptase
KW - Unnatural amino acid
UR - http://www.scopus.com/inward/record.url?scp=5344268860&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=5344268860&partnerID=8YFLogxK
U2 - 10.1016/j.pep.2004.07.019
DO - 10.1016/j.pep.2004.07.019
M3 - Article
C2 - 15477080
AN - SCOPUS:5344268860
SN - 1046-5928
VL - 38
SP - 37
EP - 44
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -