Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies
- 1 June 1993
- journal article
- research article
- Published by Springer Nature in Protein Journal
- Vol. 12 (3) , 323-327
- https://doi.org/10.1007/bf01028194
Abstract
The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofM r ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.Keywords
This publication has 14 references indexed in Scilit:
- Crystal structure of a complex of HIV‐1 protease with a dihydroxyethylene‐containing inhibitor: Comparisons with molecular modelingProtein Science, 1992
- Inhibitors of the protease from human immunodeficiency virus: design and modeling of a compound containing a dihydroxyethylene isostere insert with high binding affinity and effective antiviral activityJournal of Medicinal Chemistry, 1991
- Substrate analog inhibition and active site titration of purified recombinant HIV-1 proteaseBiochemistry, 1990
- Characterization and autoprocessing of precursor and mature forms of human immunodeficiency virus type 1 (HIV 1) protease purified from Escherichia coliProteins-Structure Function and Bioinformatics, 1989
- Recombinant HIV1 protease secreted by Saccharomyces cerevisiae correctly processes myristylated gag polyproteinProteins-Structure Function and Bioinformatics, 1989
- Genetic locus, primary structure, and chemical synthesis of human immunodeficiency virys proteaseGene Analysis Techniques, 1988
- Chemical synthesis and enzymatic activity of a 99-residue peptide with a sequence proposed for the human immunodeficiency virus protease.Proceedings of the National Academy of Sciences, 1988
- Active human immunodeficiency virus protease is required for viral infectivity.Proceedings of the National Academy of Sciences, 1988
- Enzymatic activity of a synthetic 99 residue protein corresponding to the putative HIV-1 proteaseCell, 1988
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970