Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope

FEBS Lett. 1991 Jun 3;283(2):298-302. doi: 10.1016/0014-5793(91)80613-8.

Abstract

The C-termini of p66 and p51 forms of HIV-1 reverse transcriptase have been engineered to contain a Glu-Glu-Phe sequence recognized by a monoclonal antibody to alpha-tubulin, YL1/2. Mutated RTs were purified in a single step using peptide elution from columns of immobilized YL1/2. The known sequence requirements of the YL1/2 epitope are consistent with protein eluting from the column with an intact C-terminus. Kinetic parameters of these mutated RTs are essentially unchanged from wild-type enzyme. The p15 RNaseH domain has been purified using this method and shown to have low enzyme activity compared to the parental p66 subunit.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Blotting, Western
  • Chromatography, Affinity
  • Electrophoresis, Polyacrylamide Gel
  • Endoribonucleases / genetics
  • Endoribonucleases / isolation & purification*
  • Endoribonucleases / metabolism
  • Epitopes
  • Genes, Viral
  • Genetic Engineering
  • HIV Protease / metabolism
  • HIV-1 / enzymology*
  • HIV-1 / genetics
  • Molecular Sequence Data
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Polymerase Chain Reaction
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / isolation & purification*
  • RNA-Directed DNA Polymerase / metabolism
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Restriction Mapping
  • Ribonuclease H
  • Tubulin / genetics*
  • Tubulin / immunology
  • Viral Structural Proteins / genetics

Substances

  • Epitopes
  • Recombinant Proteins
  • Tubulin
  • Viral Structural Proteins
  • RNA-Directed DNA Polymerase
  • Endoribonucleases
  • Ribonuclease H
  • HIV Protease