Date of Award

1-2014

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

John J. Siekierka

Committee Member

Jim Dyer

Committee Member

Nina M. Goodey

Subject(s)

Elephantiasis--Treatment--Research, Filarial worms, Protein kinases

Abstract

Lymphatic Filariasis (elephantiasis) is a neglected tropical disease caused by the filarial nematodes Brugia malayi (B. malayi), Brugia timori, and Wuchererici Bancrofti (1,8). These parasites are present in over 83 counties in the tropics and sub-tropics where more than 1.4 billion people are at risk of infection and 130 million people are presently infected (1,7). Previous work from our lab has led to the identification and expression of a B. malayi stress-activated protein kinase, Bm-MPKl (a human p38/C. elegans PMK-1 ortholog). Bm-MPKl plays an important role in the parasites’ protection against oxidative stress (1), and as such is a potential therapeutic drug target for the treatment of Lymphatic Filariasis. Bm-MPKl was successfully expressed in mammalian HEK-293 F cells yielding levels suitable for characterization of the enzyme. However, these cells did not produce adequate levels of recombinant protein for crystallography and high throughput screening purposes. In order to produce high levels of recombinant Bm- MPKl, I have established a novel SF9 cell/Baculovirus expression system. A synthetic Bm-MPKl was successfully subcloned into a pFASTBAC plasmid, which was then transposed into DHlOBac cells to generate a Bm-MPKl/pFASTBAC-based baculovirus. Using this system, I have demonstrated that recombinant Bm-MPKl protein kinase is highly expressed in infected SF9 insect cells. Similar to mammalian cell expression, I demonstrated that Bm-MPKl can be activated in SF9 infected cells by treatment with sodium arsenate. An excellent yield of recombinant Bm-MPKl was obtained with approximately 3.25 mg of protein recovered from a 100 mL culture.

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