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

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Department of Chemical Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2006

Author dc:creator

• Heinzelman, Pete, 1977-

Advisor dc:contributor.advisor

• Gregory N. Stephanopoulos.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/35091

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/35091