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GENETIC ENGINEERING OF THERMALLY SENSITIVE ELASTIN-LIKE POLYPEPTIDE AND ITS EXPRESSION IN HEK 293 CELLS
by
Sejal Parakh
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)
August 2010
Copyright 2010 Sejal Parakh
Object Description
| Title | Genetic engineering of thermally sensitive elastin-like polypeptide and its expression in HEK 293 cells |
| Author | Parakh, Sejal |
| Author email | sparakh@usc.edu; sejal.parakh@gmail.com |
| Degree | Master of Science |
| Document type | Thesis |
| Degree program | Pharmacy / Pharmaceutical Sciences |
| School | School of Pharmacy |
| Date defended/completed | 2010-06-04 |
| Date submitted | 2010 |
| Restricted until | Unrestricted |
| Date published | 2010-08-06 |
| Advisor (committee chair) | MacKay, Andrew J. |
| Advisor (committee member) |
Okamoto, Curtis Hamm-Alvarez, Sarah |
| Abstract | Substantial efforts have been to design drug carriers for targeted drug delivery to tumor sites in order to spare the normal tissue from the hazardous effects of chemotherapeutic agents and achieve better therapeutic efficacy. One promising approach to reduce chemotoxicity would be to conjugate or encapsulate these potent drugs into nanometer (10-400) sized particles and trigger their release at tumor sites. However, nanoparticulates frequently accumulate in the liver, which leads to high local concentrations of these toxic drugs. This accumulation reduces delivery of the appropriate dose of drugs to the site of action and can generate hepatotoxicity.; The aim of this project is to design a nano drug carrier that extends plasma circulation, permits greater tumor accumulation, and reduces hepatotoxicity, which is based on the enhanced permeability and retention effect. To achieve this goal, a novel approach to produce N-glycosylated peptide nanoparticles has been explored.; Elastin-like polypeptides are repeated pentameric peptides VPGXG exhibiting a characteristic inverse phase transition temperature Tt, above which they form aggregates from a monodisperse solution. It is hypothesized that on triggering the phase transition of an N-glycosylated-ELP-drug conjugate, it will form nanostructures with insoluble ELP-drug at the core shielded with soluble N-linked carbohydrates. For this purpose, a library of ELP VPGAG consisting of 6, 12, 24, 48, 96, 192 pentamers was constructed. This was followed by biosynthesis of several constructs comprising of ELP A96 and A192 with Endoplasmic Reticulum Signal and N-glycosylation sequences using recombinant DNA technology.; Expression of these constructs and GFP plasmid was optimized into mammalian HEK 293 cells using L-PEI as transfection agent. Successful expression of GFP protein alone and its co-transfection with N-glycosylation + ELP A192 and A96 sequences was evaluated using fluorescence microscopy. However, N-glycosylated ELP or ELP (A192 and A96) did not exhibit any obvious phase transition in the medium collected before and after HEK293 cell lysis. Thus, expression of N-glycosylated ELP or ELP protein could not be determined. |
| Keyword | elastin-like polypeptide; recursive directional ligation; inverse transition cycling; N-glycosylation |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m3329 |
| Rights | Parakh, Sejal |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | http://www.usc.edu/isd/libraries/services/ask_a_librarian/email/ |
| Filename | etd-Parakh-3976 |
| Archival file | uscthesesreloadpub_Volume48/etd-Parakh-3976.pdf |
Description
| Title | Page 1 |
| Full text | GENETIC ENGINEERING OF THERMALLY SENSITIVE ELASTIN-LIKE POLYPEPTIDE AND ITS EXPRESSION IN HEK 293 CELLS by Sejal Parakh A Thesis Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (PHARMACEUTICAL SCIENCES) August 2010 Copyright 2010 Sejal Parakh |
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