title: Heterogeneity in recombinant HIV-1 integrase corrected by site-directed mutagenesis: the identification and elimination of a protease cleavage site.
abstract: Purified recombinant human immunodeficiency virus type 1 (HIV-1) integrase and certain deletion mutants exhibit heterogeneity consistent with proteolysis at a site close to the C-terminus. Electrospray ionization mass spectrometric analysis indicated that proteolytic cleavage generated a protein missing five residues from the C-terminus. PCR mutagenesis of amino acids on either side of the cleavage site identified two changes which were subsequently shown to prevent clipping when proteins were expressed and purified from Escherichia coli: the substitution of Arg284, the residue on the C-terminal side of the cleavage site, by either glycine or lysine. The introduction of either of these mutations into full-length integrase did not affect in vitro 3' processing or strand transfer activities. Thus, the incorporation of either of these mutations is likely to be beneficial when homogeneity of HIV-1 integrase is a concern, as in crystallographic or nuclear magnetic resonance spectroscopic experiments.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: A B
authors.initials: AB
authors.lastname: Hickman
authors.forename: F
authors.initials: F
authors.lastname: Dyda
authors.forename: R
authors.initials: R
authors.lastname: Craigie
mesh_terms: D001665:Binding Sites / Q000235:genetics; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray; D004926:Escherichia coli; D019427:HIV Integrase / Q000235:genetics / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
chemical_list: D019427:HIV Integrase; D016333:HIV Protease