Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Design and synthesis of aryl hydrocarbon receptor fusion proteins for polyclonal antibodies production and cellular delivery

Author

Bhagyashree Yogesh Bhagwat, University of the Pacific

Date of Award

2001

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutics and Medicinal Chemistry

First Advisor

William K. Chan

First Committee Member

Xiaoling Li

Second Committee Member

Timothy Smith

Abstract

Polycyclic aromatic hydrocarbons are environmental chemicals that are produced during incomplete combustion of coal, oil, gas, and garbage. Toxic effects of these compounds are mediated via the ligand activated Aryl Hydrocarbon Receptor (AHR) signaling pathway. To enable the study of the AHR signaling mechanism, our lab has generated many human proteins using recombinant DNA technology. This thesis documents the design and synthesis of a number of proteins of the AHR deletion construct CΔ553. The bacterial expressed and purified fusion proteins could be utilized as antigen to generate antibodies and be used for cellular delivery. The purified protein was immunogenic in rabbits and produced significant amount of polyclonal antibodies. In western blot analysis, the antibodies were able to the detect baculovirus expressed AHR and different recombinant proteins of the AHR. The polyclonal antibodies were also used in the gel-shift assay to show the AHR dependent gel shift. Cellular delivery CΔ553 was achieved using the protein transduction domain from the HIV-1 virus transactivating protein (TAT). In order to deliver the CΔ553 into mammalian cells, an expression vector was constructed to generate the TAT-CΔ553 fusion protein. The TAT-CΔ553 fusion protein was successfully transduced into two mammalian cells-HeLa and HepG2. The in vivo function of TAT-CΔ553 was determined using the luciferase reporter plasmid assay. The transduced protein was functional; it competed with the AHR and heterodimerize with ARNT in both HeLa and HepG2 cells at a concentration of 3.8x103 nM and 18 nM respectively. Since there an apparent similarity between the basic region of TAT-PTD and CΔ553, we examined the transduction potential of CΔ553. Western blot analysis indicated that the extent of denatured protein transduction was comparable for CΔ553 and TAT-CΔ553 in HepG2 cells. Thus CΔ553 might have intrinsic transduction capability.

Pages

87

Recommended Citation

Bhagwat, Bhagyashree Yogesh. (2001). Design and synthesis of aryl hydrocarbon receptor fusion proteins for polyclonal antibodies production and cellular delivery. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/558