Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence

Abstract

We have developed the first protease specificity engineering technology intended to serve as a general-purpose source of target-specific protease mutants with potential therapeutic applications. We used this E. coli-based screening system to isolate multiple HIV protease mutants with improved proteolytic specificities and activities toward an octapeptide taken from the beta-amyloid peptide (Abeta) implicated in Alzheimer's Disease. Quantitative Western blotting of E. coli extracts revealed approximately 20- and 30-fold increases in proteolytic specificity and activity, respectively, toward the Abeta octapeptide relative to a native octameric substrate of the wild type HIV protease. Our HIV protease specificity engineering system requires no expensive reagents or equipment and can be conveniently adopted by any laboratory. The system employs the E. coli betagalactosidase protein as a reporter for protease activity. The employment of Pgal as a reporter gene allows facile blue/white E. coli colony screening of mutant protease libraries. Additionally, mutant protease specificity profiles are readily assessed via E. coli liquid culture Pgal activity assays.

(cont.) Mutant protease library screening stringency is conveniently controlled by the amount of arabinose added to blue/white screening plates. We have identified Alzheimer's Disease, Parkinson's Disease, HIV and Hepatitis C as maladies that may be treated with target-specific proteases obtained by using our system. We are eager to observe the future pursuit of both these and additional biomedical applications for target-specific proteases.