Protein Production, Analysis and Assay Development

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The purpose of the Core is to prepare, purify, and test all recombinant, wild type and drug resistant proteases and prepare polypeptide and fluorigenic substrates required for the overall Program Project. This Core will provide the reagents and background testing required of all the projects, including studies of substrate/inhibitor specificity (Olson, Torbett), inhibitor development (Finn/Sharpless, Stout/McCree) and structural analyses of drug-resistant proteases (Stout Core/Torbett). Additionally, the Protein Expression Core will interact directly with the other Cores to characterize predominant drug resistant proteases revealed in analyses of patient samples (Looney) and provide enzymes and substrates required for crystallographic analyses (Stout). In the previous years of this ongoing and productive collaboration, we have prepared and purified over 50 wild type, drug-resistant, and mutant proteases from HIV-1, FIV, and SIV, each cloned and over-expressed in E. coli. These constructs will be maintained for use by the program and the repertoire will be expanded as more sequences become available. Highly purified proteases will be prepared and supplied as detailed below. In addition, new proteases based on structural changes associated with drug resistance that are identified by the projects will be prepared for analysis of relative Kcat and Km values as well as to assess the relative Ki of various inhibitors in the context of these proteases. The Core will also continue to develop additional substrates from regions of the Gag-Pol polyprotein, including the primary cleavage sites on either side of NC as well as seven other cleavage sites in Gag-Pol. Where applicable, ex vivo tissue culture analyses of inhibitor efficacy and specificity will be carried out, primarily using a pseudovirion expression system developed by this Core that allows assessment of efficacy of PR cleavage of Gag and Gag/Pol polyproteins, as well as inhibitor potency, specificity and cell toxicity. Tests of efficacy against infectious HIV-1 will be performed in BSL-3 facilities under the direction of the Torbett component.