Event Title

Expression, Purification, and Crystallization of an Endoxylanase from Bacteroides vulgatus

Faculty Mentor

Dr. Jason Hurlbert

College

College of Arts and Sciences

Department

Chemistry, Physics and Geology

Location

DiGiorgio Campus Center, Room 221

Start Date

22-4-2016 2:45 PM

End Date

22-4-2016 3:00 PM

Description

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)

Comments

McNair Scholar

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Apr 22nd, 2:45 PM Apr 22nd, 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.