Single-step purification of recombinant wild-type and mutant HIV-1 reverse transcriptase

Protein Expr Purif. 1996 Feb;7(1):27-32. doi: 10.1006/prep.1996.0004.


We have devised a single-step method that enables purification of HIV-1 recombinant reverse transcriptase directly from bacterial lysates in less than 2 h. Clarified lysates are applied to commercial Q- and S-matrix cartridge columns connected in series. The columns are washed with low-salt buffer to remove unbound protein, then the Q column is removed and reverse transcriptase is eluted from the S column using a salt gradient. The purification has been carried out with both medium-pressure and high-pressure chromatographic systems. Purifications are carried out at room temperature near neutral pH, providing enzyme with high DNA polymerase specific activity. A crucial aspect of the procedure is the use of Tris buffer, a buffer that is normally incompatible in cation-exchange methods. The method is applicable for the purification of the p51/p66 heterodimer and the p5l and p66 homodimer forms of reverse transcriptase. We have used this method to purify wild-type reverse transcriptase and several recombinant proteins containing mutations correlated with dideoxynucleoside drug resistance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blotting, Western
  • Chromatography, High Pressure Liquid
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyguanine Nucleotides / metabolism
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • HIV Reverse Transcriptase / chemistry
  • HIV Reverse Transcriptase / genetics
  • HIV Reverse Transcriptase / isolation & purification*
  • HIV Reverse Transcriptase / metabolism
  • Isoenzymes / chemistry
  • Isoenzymes / genetics
  • Isoenzymes / metabolism
  • Kinetics
  • Molecular Weight
  • Mutation
  • Oligodeoxyribonucleotides / metabolism
  • Poly A / metabolism
  • Polymerase Chain Reaction
  • Protein Conformation
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Thymine Nucleotides / metabolism


  • Deoxyguanine Nucleotides
  • Isoenzymes
  • Oligodeoxyribonucleotides
  • Recombinant Proteins
  • Thymine Nucleotides
  • Poly A
  • poly(rA).oligo(dT)
  • deoxyguanosine triphosphate
  • HIV Reverse Transcriptase
  • DNA-Directed DNA Polymerase
  • thymidine 5'-triphosphate