| Autor: |
Stahlhut, M., Li, Y., Condra, J.H., Fu, J., Gotlib, L., Graham, D.J., Olsen, D.B. |
| Zdroj: |
Protein Expression and Purification; December 1994, Vol. 5 Issue: 6 p614-621, 8p |
| Abstrakt: |
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.Copyright 1994, 1999 Academic Press, Inc. |
| Databáze: |
Supplemental Index |
| Externí odkaz: |
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