Date of Award

2017

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Administrative Home Department

Department of Chemistry

Advisor 1

Martin Thompson

Committee Member 1

Caryn Heldt

Committee Member 2

Tarun Dam

Committee Member 3

Marina Tanasova

Abstract

Over the past decade, it has become apparent that the human polybromo-1 protein (BAF180) has a critical role in cancer. BAF180 is known to be a driver mutation in clear cell renal cell carcinoma, where it has been found to be mutated in approximately 40% of cases. Mutations have also been found in several other cancers, including intrahepatic cholangiocarcinomas and epithelioid sarcomas. BAF180 is the chromatin targeting subunit of the PBAF (Polybromo-associated BRG1-associated factor) chromatin remodeling complex, a role facilitated by its nine domains: six bromodomains, which recognize and bind to acetylated lysines on histones; two BAH (bromo-adjacent homology) domains, found to be critical for PCNA ubiquitination following DNA damage; and one HMG (high mobility group) box, the DNA binding component. Furthermore, proper expression of BAF180 has also been linked to cardiac development and cell cycle regulation.

Despite these associations, the molecular level interactions of full-length BAF180 have yet to be studied; only the phenomenological effects of BAF180 deficiency/mutation have been studied. It is crucial that we understand the binding dynamics and specificities of wild type and mutated BAF180, since it is the recognition component of PBAF.

Expression of the recombinant full-length BAF180 protein has been difficult because of the complex nature of this protein, its unusual codon usage, and size. After E. coli expression failed, other expression systems were investigated and the yeast Pichia pastoris was chosen. Pichia was chosen for several reasons: its codon usage is similar to that of BAF180 and it is a eukaryotic system possessing eukaryotic protein folding machinery and capable of performing post-translational modifications. Under control of the GAP promoter, full-length BAF180 has been successfully expressed in Pichia pastoris. This is the first time that full-length BAF180 has been cloned and expressed in a heterologous host. It was purified using anion exchange chromatography.

The ability to express and purify full-length BAF180 is a huge first step towards increasing the understanding of the molecular mechanisms of this protein and its association with cancer development.

Available for download on Tuesday, May 01, 2018

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