Cloning, Expression and Purification of the Antimicrobial Targets EtfB and EtfDh of Burkholderia cenocepacia

Abstract

Burkholderia cenocepacia is a pathogenic bacterium that causes life-threatening infections in cystic fibrosis patients (Shommu, Vogel, & Storey, 2015). Through a conditional growth mutant library, two essential genes of this bacterium, etfB and etfDh, encoding an electron transfer flavoprotein unit and an etf dehydrogenase respectively, have been identified. While the essential role of these proteins in B. cenocepacia is unknown, protein characterization and interaction analysis will aid in the development of their capacity as antimicrobial targets. The experimental goal of this study was to clone, express and purify etfB and etfDh. Both genes were separately cloned into the bacterial plasmid pE-SUMO and transformed into E. coli BL21-DE3 GOLD. Protein expression of EtfB and EtfDh was induced by isopropyl β-D-1-thiogalactopyranoside (IPTG), and the soluble and insoluble protein fractions were analyzed. EtfB was found in the soluble fraction, which is expected as ETFs are cytoplasmic proteins (Winsor, Khaira, Van Rossum, Lo, Whiteside, & Brinkman, 2008). EtfDh was mainly present in the insoluble fraction suggesting that the B. cenocepacia EtfDh is a membrane-bound dehydrogenase (Winsor et al., 2008). The protein purification process has resulted in a highly pure EtfB and EtfDh samples, which can be used to raise antibodies. Consequently, antibodies raised against these proteins will allow immunofluorescence and coimmunoprecipitation studies of EtfB and EtfDh. The results of the current work provide the tools to address the hypothesis of an intracellular interaction between cytoplasmic EtfB and membrane-bound EtfDh to further understand their role and essentiality in B. cenocepacia.

Author Biography

Allison L. Balasko, University of Manitoba
Department of Microbiology, Undergraduate Researcher
Published
2016-12-24
Section
Articles