Expression, Purification, and Crystallization of an Endoxylanase from Bacteroides vulgatus
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Faculty Mentor
Dr. Jason Hurlbert
Abstract
Demand for sustainable energy sources is growing as fossil fuels are rapidly expended. One such source is fuel ethanol produced via fermentation of plant biomass generated by engineered biocatalysts. The creation of such biocatalysts requires identifying novel enzymes capable of degrading specific carbohydrate polymers and cloning these enzymes into a bacterial host for structural characterization. This study seeks to characterize a novel endoxylanase of glycosyl hydrolase family 30 (GH30) from Bacteroides vulgatus, a bacterium found in the human gastrointestinal microbiome, via x-ray crystallography. The gene for B. vulgatus GH30 endoxylanase (BvGH30) was cloned into a pET 28b prokaryotic expression vector which was used to transform a culture of Escherichia coli. The resulting bacterial strain expressed the cloned BvGH30, and the recombinant protein produced was purified to homogeneity via Ni2+-Metal Chelating Affinity Chromatography (MCAC) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was concentrated to 10 mg/mL and used to screen for solution conditions that promoted crystal growth via sparse matrix screening in hanging drop, vapor-diffusion plates. Single cubic crystals of defined morphology >0.1mm in length were obtained in 20% w/v PEG 3350, 0.2M NaF. Single rectangular crystals <0.1mm in length were found in 0.2M (NH4)2HPO4, 20% (w/v) PEG 3350, pH 8.0, and large numbers of smaller rectangular crystals were obtained in 0.1M Citric acid, 0.8M (NH4)2SO4, pH 4.0. Grid screening around these conditions will be employed to optimize crystal size and morphology, and crystals greater than 0.1mm in length will be subjected to x-ray diffraction analysis.
Previously Presented/Performed?
SAEOPP McNair/SSS Scholars Research Conference, Atlanta, Georgia, June 2015
Summer Undergraduate Research Experience (SURE) Poster Session, Winthrop University, September 2015
Grant Support?
Supported by a grant from the National Institutes of Health IDeA Networks for Biomedical Research Excellence (NIH INBRE)
Start Date
22-4-2016 2:45 PM
End Date
22-4-2016 3:00 PM
Expression, Purification, and Crystallization of an Endoxylanase from Bacteroides vulgatus
DiGiorgio Campus Center, Room 221
Demand for sustainable energy sources is growing as fossil fuels are rapidly expended. One such source is fuel ethanol produced via fermentation of plant biomass generated by engineered biocatalysts. The creation of such biocatalysts requires identifying novel enzymes capable of degrading specific carbohydrate polymers and cloning these enzymes into a bacterial host for structural characterization. This study seeks to characterize a novel endoxylanase of glycosyl hydrolase family 30 (GH30) from Bacteroides vulgatus, a bacterium found in the human gastrointestinal microbiome, via x-ray crystallography. The gene for B. vulgatus GH30 endoxylanase (BvGH30) was cloned into a pET 28b prokaryotic expression vector which was used to transform a culture of Escherichia coli. The resulting bacterial strain expressed the cloned BvGH30, and the recombinant protein produced was purified to homogeneity via Ni2+-Metal Chelating Affinity Chromatography (MCAC) as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein was concentrated to 10 mg/mL and used to screen for solution conditions that promoted crystal growth via sparse matrix screening in hanging drop, vapor-diffusion plates. Single cubic crystals of defined morphology >0.1mm in length were obtained in 20% w/v PEG 3350, 0.2M NaF. Single rectangular crystals <0.1mm in length were found in 0.2M (NH4)2HPO4, 20% (w/v) PEG 3350, pH 8.0, and large numbers of smaller rectangular crystals were obtained in 0.1M Citric acid, 0.8M (NH4)2SO4, pH 4.0. Grid screening around these conditions will be employed to optimize crystal size and morphology, and crystals greater than 0.1mm in length will be subjected to x-ray diffraction analysis.
