Abstract

Throughout history, Mycobacterium tuberculosis (MTB) has killed more people than any other infectious agent, while Mycobacterium leprae is an ancient human pathogen that still causes disease today. Each has developed drug resistance, thus the discovery of potential novel antibiotic targets is essential. The diadenosine polyphosphatases (ApnAases) / mRNA decapping enzymes of the Nudix hydrolase superfamily have been found to be essential for a number of pathogens (Bartonella bacilliformis, Legionella pneumophila) to invade human host cells, a process often essential for pathogenesis, and thus potential novel drug targets. There are two ApnAases in MTB: Rv2985 and Rv3908 and two corresponding orthologs in M. leprae: Ml1682 and Ml2698. Our laboratory has cloned, expressed, purified, and characterized Rv2985. Here, I describe the subcloning, expression, and partial purification of Rv3908, Ml1682, and Ml2698. The three genes were each subcloned to incorporate a His•Tag onto the N-terminus of each protein. Each His•Tagged protein was expressed and purified via Ni2+ affinity chromatography. The two ApnAases from M. leprae are fairly insoluble and thus a number of experiments were carried out aimed at increasing Ml1682’s solubility: varying expression temperature, Isopropyl-β-D-thiogalactopyranoside (IPTG) concentration, or time of expression after induction; the use of the chaperone GroESL, codon+ E. coli strain Rosetta(DE3), minimal media; or inclusion of glycylglycine. These standard methods of solubilization did not improve the solubility of this ApnAase from M. leprae, and thus other methods to improve solubility will need to be determined before we purify and characterize these enzymes. Rv3908 is more soluble, and is ready to purify and characterize.

Library of Congress Subject Headings

Hydrolases--Analysis; Drug targeting; Drug resistance in microorganisms

Publication Date

6-2015

Document Type

Thesis

Student Type

Graduate

Degree Name

Chemistry (MS)

Department, Program, or Center

School of Chemistry and Materials Science (COS)

Advisor

Suzanne O’Handley

Advisor/Committee Member

Austin Gehret

Advisor/Committee Member

André O. Hudson

Comments

Physical copy available from RIT's Wallace Library at PQ609.P5 Z48 2015

Campus

RIT – Main Campus

Plan Codes

CHEM-MS

Download

Share

COinS