| Popis: |
Recently, we reported [1] a single step, large scale purification of recombinant HIV protease, wherein Escherichia coli inclusion bodies harboring the enzyme are dissolved in 50% acetic acid and the solution is subjected to size exclusion chromatography in the same solvent on a column of Sephadex G-75. This was an important advance in our studies of the protease because it afforded large quantities of highly pure and active enzyme in a short time. However, we still encountered difficulties because of autodigestion at the three sites (Leu5-Trp6, Leu33-Glu34 and Leu63-Ile64) [1,2,3] which took place during the concentration process, repeated freeze-thawing, and prolonged storage. Since much of our research had been devoted to understanding the specificity of the HIV-1 and 2 proteases, we knew a good deal about what amino acid substitutions could be made in a peptide sequence in order to destroy its susceptibility to hydrolysis. These ideas were incorporated into site-directed mutagenesis of the HIV-1 protease gene to make a variety of HIV-1 protease mutants that are superior with respect to the wild-type enzyme in terms of stability to autodigestion and yet which maintain the same substrate specificity. This paper deals with the technology employed for the design and expression of HIV-1 protease mutants, their purification, and the refolding of inactive proteins to active enzymes. A companion paper [4] describes in some detail the strategy we used to select certain mutations at the P1 and/or P2’ position to obtain mutants resistant to autolysis, and the effects of the mutations on the enzyme’s stability and activity. |
