Publication Date

1998

Document Type

Dissertation/Thesis

First Advisor

Mitchell, John L. A.

Degree Name

M.S. (Master of Science)

Legacy Department

Department of Biological Sciences

LCSH

Hepatoma--Genetic aspects; Ornithine decarboxylase--Inhibitors; Chemical inhibitors--Genetic aspects; Antineoplastic agents--Genetic aspects; Proteins--Genetic aspects

Abstract

Antizyme is a novel regulatory protein that has two distinct functions that result in cellular polyamine suppression. Cells contain a protein named antizyme inhibitor (Ain) for its ability to bind antizyme with high affinity, and effect the release and reactivation of bound ornithine decarboxylase (ODC). Although antizyme inhibitor is highly homologous to ODC, its physiological function remains poorly understood. Acquisition of the genetic information encoding antizyme inhibitor is essential to designing experiments that may elucidate its role in polyamine homeostasis. This report describes the cloning of antizyme inhibitor cDNA from a mammalian cell line, and the expression and purification of this protein. The cDNA was cloned from HTC rat hepatoma cells into the bacterial expression vector pGEX-3X, and the 1344 bp open reading frame was completely sequenced. The sequence was identical to the reported sequence for rat heart Ain except for a conservative amino acid difference at residue 91. The Ain protein was expressed in E.Coli as a glutathione S-transferase fusion construct under control of an IPTG-inducible promotor. The protein was solubilized with 8M urea from inclusion bodies and purified using ion-exchange chromatography. This material was used to inject a rabbit for production of antisera to antizyme inhibitor. The cDNA and antibody are powerful tools to be used for the investigation of the physiological role of antizyme inhibitor.

Comments

Includes bibliographical references (pages 70-74)

Extent

iv, 74 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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