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Date of Award

1997

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

William K. Chan

First Committee Member

Gregg D. Jongeward

Second Committee Member

Craig Vierra

Abstract

The DNA- binding complex Hypoxia-inducible factor 1 (HIF-1), a heterodimeric basic helix-loop-helix (bHLH) transcription factor, is involved in the regulation of gene expression by oxygen, a prime target being the human erythropoietin (EPO) gene. In this study, the human HIF-1α subunit was expressed in a baculovirus system. To simplify purification, a polyhistidine tag was attached to the C terminus to facilitate the fusion protein to be specifically adsorbed to a Talon metal affinity resin.

Expression studies revealed that this overexpressed HIF-1α was found in soluble, nuclear, and insoluble cell extracts. Electrophoretic mobility shift assays (EMSA) demonstrated that the fusion protein heterodimerize with the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT), and bound to a probe spanning nucleotides 1 to 18 (W18) of the human erythropoietin gene enhancer that confers hypoxic induction. This study also demonstrates that the presence of 7 mM hemin or different cobalt concentrations (50 mM; 100 mM; and 200 mM) results in no obvious effect on the solubility or expression level of HIF-1a in insect cells. Similarly, coexpression of ARNT and HIF-1α in Sf9 cells did not increase solubility of the recombinant HIF-1α protein.

Gel shift assays demonstrated that the Talon-purified recombinant HIF-1α and ARNT proteins could bind the W18 DNA probe only when reconstituted in the presence of a heat-sensitive factor(s) found in soluble and nuclear extracts of Sf9 cells as well as in rabbit reticulocyte lysate. Interestingly, reconstitution of DNA binding activity was only possible when using HIF-1α purified under native conditions. When using HIF-1α purified under denaturing conditions and refolded, DNA-binding activity was absent.

Pages

58

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