Purification and Characterization of HIV-1 Reverse Transcriptase Having a 1:1 Ratio of p66 and p51 Subunits
Identifieur interne : 004406 ( Main/Exploration ); précédent : 004405; suivant : 004407Purification and Characterization of HIV-1 Reverse Transcriptase Having a 1:1 Ratio of p66 and p51 Subunits
Auteurs : M. Stahlhut [États-Unis] ; Y. Li [États-Unis] ; J. H. Condra [États-Unis] ; J. Fu [États-Unis] ; L. Gotlib [États-Unis] ; D. J. Graham [États-Unis] ; D. B. Olsen [États-Unis]Source :
- Protein Expression and Purification [ 1046-5928 ] ; 1994.
Abstract
Abstract: Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 Å resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.
Url:
DOI: 10.1006/prep.1994.1084
Affiliations:
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Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 Å resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.</div></front></TEI><affiliations><list><country><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Stahlhut, M" sort="Stahlhut, M" uniqKey="Stahlhut M" first="M." last="Stahlhut">M. Stahlhut</name></noRegion><name sortKey="Condra, J H" sort="Condra, J H" uniqKey="Condra J" first="J. H." last="Condra">J. H. Condra</name><name sortKey="Fu, J" sort="Fu, J" uniqKey="Fu J" first="J." last="Fu">J. Fu</name><name sortKey="Gotlib, L" sort="Gotlib, L" uniqKey="Gotlib L" first="L." last="Gotlib">L. Gotlib</name><name sortKey="Graham, D J" sort="Graham, D J" uniqKey="Graham D" first="D. J." last="Graham">D. J. Graham</name><name sortKey="Li, Y" sort="Li, Y" uniqKey="Li Y" first="Y." last="Li">Y. Li</name><name sortKey="Olsen, D B" sort="Olsen, D B" uniqKey="Olsen D" first="D. B." last="Olsen">D. B. Olsen</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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