Date of Award

Winter 1-27-2017

Degree Type

Thesis

Degree Name

MS Microbiology

Department

Biology

Advisor

Daniel B. Nichols, Ph.D

Committee Member

Cosimo Antonacci, Ph.D

Committee Member

Edward Tall, Ph.D

Committee Member

Angela Klaus, Ph.D

Committee Member

Heping Zhou, Ph.D

Keywords

Poxvirus, Purification, Vaccinia, Transcription, A7, D6

Abstract

The eradication of Smallpox is one of the greatest human achievements. However other poxviruses still exist infecting humans (Molluscum Contagiosum, monkeypox, and Vaccinia) or livestock (cowpox, sheeppox). Though vaccines and anti-virals exist, the side effects are serious, and viral resistance has been detected. The genome amongst poxviruses is highly conserved, early gene promoters maintain a specific critical consensus region that can be targeted for development of novel broad spectrum therapeutics. Using Vaccinia early transcription factors (VETFs) a novel method of expressing and purifying VETF A7 and D6 was developed. A7 and D6 are required for transcription of early genes recruiting the RNA polymerase to the site of transcription. Cloning of A7L and D6R into the bacterial expression vector pET302/303 N-terminus/C-terminus His-Tag was done in order to express A7 and D6 in the Escherichia coli (E. coli) DH5a and BL21 DE3 strains. Under Isopropyl b-D-1-thiogalactopyranoside (IPTG) induction, BL21s can be controlled to express proteins under time courses and different concentrations of the inducer. Optimizing provided a method where concentration of VETFs A7 and D6 expression was controlled but abundant. After, different methods of extraction were used to solubilize A7 in order for purification to be done. Nonionic buffers were not strong enough to solubilize the majority of A7 from samples, however anionic buffers are able to extract the majority of the VETF. Purification of A7 was successful but further optimization is required, and although D6 was successfully solubilized purification still has to be done. This novel system of expression and purification using bacteria is the first known attempt to purify VETFs A7 and D6, and is significantly cheaper and safer than known methods. Thus, this system opens the door for a cost effective and safer method for future studies.

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