Abstract 1866 Structural Characterization of TonB3 (BT1668) from Bacteroides thetaiotaomicron

Starch is digested in the human body by bacteria in the gut microbiome. Bacteroides thetaiotaomicron (B. theta) exists in the large intestine and helps the body break down starch via the starch utilization system (Sus). Within this outer membrane complex, SusC is designated as a TonB-dependent transporter (TBDT) which features a conserved motif known as the Ton Box. The starch molecule binds to SusC leading to a reaction between the Ton Box and the inner membrane protein TonB. This opens a channel in SusC, allowing starch to enter the cell. B. theta encodes 11 TonB homologs that could pair with SusC and other TBDTs. After screening several homologs for robust expression in Escherichia coli we have chosen to focus on TonB3 (BT1668). The goal of this work is to express the TonB3 in E. coli cells and then purify it using fast protein liquid chromatography (FPLC). We successfully cloned the genetic region encoding for the C-terminus of TonB3 into the pETite expression plasmid. Sequencing confirmed the formation of the plasmid containing the TonB3 gene. The plasmid was then successfully inserted into Rosetta pLysS cells. Attempts to purify TonB3 using His-purification alone were not sufficient, so we are testing ion exchange and gel filtration to increase purification. Future work includes determining the protein structure with x-ray crystallography and conducting interaction studies between TonB3 and SusC.