Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography

Eur J Biochem. 1990 Jan 26;187(2):307-14. doi: 10.1111/j.1432-1033.1990.tb15306.x.


We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.

MeSH terms

  • Chelating Agents
  • Chromatography, Affinity
  • Cloning, Molecular
  • Electrophoresis, Polyacrylamide Gel
  • Endoribonucleases / genetics
  • Endoribonucleases / isolation & purification
  • Escherichia coli / metabolism
  • Gene Expression Regulation, Enzymologic*
  • Gene Products, pol / isolation & purification
  • Genetic Vectors
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Humans
  • Plasmids
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / isolation & purification*
  • Ribonuclease H


  • Chelating Agents
  • Gene Products, pol
  • RNA-Directed DNA Polymerase
  • Endoribonucleases
  • Ribonuclease H