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