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Rapid purification and characterisation of HIV‐1 reverse transcriptase and RNaseH engineered to incorporate a C‐terminal tripeptide α‐tubulin epitope
Author(s) -
Stammers D.K.,
Tisdale M.,
Court S.,
Parmar V.,
Bradley C.,
Ross C.K.
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80613-8
Subject(s) - reverse transcriptase , epitope , rnase h , tripeptide , microbiology and biotechnology , rna directed dna polymerase , peptide , protein subunit , enzyme , biology , chemistry , monoclonal antibody , biochemistry , antibody , rna , genetics , gene
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 α‐tubulin YL . Mututed RTs were purified in a single step using peptide elution from columns of immobilized YL . The known sequence requirements of the YL epitope arc 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.