Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006.
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| Format: | Thesis |
| Language: | eng |
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Massachusetts Institute of Technology
2006
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| Online Access: | http://hdl.handle.net/1721.1/35091 |
| _version_ | 1811069987698245632 |
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| author | Heinzelman, Pete, 1977- |
| author2 | Gregory N. Stephanopoulos. |
| author_facet | Gregory N. Stephanopoulos. Heinzelman, Pete, 1977- |
| author_sort | Heinzelman, Pete, 1977- |
| collection | MIT |
| description | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006. |
| first_indexed | 2024-09-23T08:20:06Z |
| format | Thesis |
| id | mit-1721.1/35091 |
| institution | Massachusetts Institute of Technology |
| language | eng |
| last_indexed | 2024-09-23T08:20:06Z |
| publishDate | 2006 |
| publisher | Massachusetts Institute of Technology |
| record_format | dspace |
| spelling | mit-1721.1/350912022-01-13T07:54:19Z Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence Heinzelman, Pete, 1977- Gregory N. Stephanopoulos. Massachusetts Institute of Technology. Dept. of Chemical Engineering. Massachusetts Institute of Technology. Department of Chemical Engineering Chemical Engineering. Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006. Includes bibliographical references (leaves 119-121). 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. by Pete Heinzelman. Ph.D. 2006-12-18T20:39:29Z 2006-12-18T20:39:29Z 2006 2006 Thesis http://hdl.handle.net/1721.1/35091 71332527 eng 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. http://dspace.mit.edu/handle/1721.1/7582 121 leaves 17611598 bytes 17611099 bytes application/pdf application/pdf application/pdf Massachusetts Institute of Technology |
| spellingShingle | Chemical Engineering. Heinzelman, Pete, 1977- Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title | Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title_full | Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title_fullStr | Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title_full_unstemmed | Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title_short | Identification of HIV protease mutants with improved specificities toward an Alzheimer's Disease associated peptide sequence |
| title_sort | identification of hiv protease mutants with improved specificities toward an alzheimer s disease associated peptide sequence |
| topic | Chemical Engineering. |
| url | http://hdl.handle.net/1721.1/35091 |
| work_keys_str_mv | AT heinzelmanpete1977 identificationofhivproteasemutantswithimprovedspecificitiestowardanalzheimersdiseaseassociatedpeptidesequence |