Rapid purification and characterisation of HIV-1 reverse transcriptase and RNaseH engineered to incorporate a C-terminal tripeptide alpha-tubulin epitope.
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.
Stammers
D K
DK
Department of Molecular Sciences, Wellcome Research Laboratories, Beckenham, Kent, UK.
Tisdale
M
M
Court
S
S
Parmar
V
V
Bradley
C
C
Ross
C K
CK
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000939:Epitopes; D005814:Genes, Viral; D005818:Genetic Engineering; D016333:HIV Protease / Q000378:metabolism; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016914:Ribonuclease H; D014404:Tubulin / Q000235:genetics* / Q000276:immunology; D015678:Viral Structural Proteins / Q000235:genetics
D000939:Epitopes; D011994:Recombinant Proteins; D014404:Tubulin; D015678:Viral Structural Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H; D016333:HIV Protease