Date of Award

8-2013

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Jana L. Villemain, Ph.D.

Second Advisor

John C. Ford, Ph.D.

Third Advisor

Keith S. Kyler, Ph.D.

Fourth Advisor

Jonathan N. Southard, Ph.D.

Abstract

This thesis examines the beginning steps of the investigation into possible conformational changes in the C-terminus of the Srs2 helicase. This portion of the protein was predicted, using bioinformatics programs, like GlobPlot1 and IUpred2, to possess characteristics related to the class of proteins known as intrinsically unstructured proteins. Initially, the full length Srs2 protein was expressed in BL21 (DE3) (Rosetta 2) cells and preliminary steps were taken to isolate it using an Ni-NTA column. Next, a set of bacterial transcription factors were expressed and subsequently isolated using an Ni-NTA column. To produce C-terminal portions of Srs2, two inserts were cloned separately into the pET 28a+ plasmid. These plasmids were then transferred into a variety of cell lines. This includes the DH5alpha; (plasmid isolation) and Rosetta 2 (protein expression) cells. After expression optimization, several protein purification trials were conducted.

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