Event Title

Expression, Purification and Preliminary Crystallization of a Putative Chaperonin from Xanthomonas cynarae

Poster Number

037

College

College of Arts and Sciences

Department

Department of Chemistry, Physics, and Geology

Honors Thesis Committee

Jason Hurlbert, Ph.D.; Fatima Amir, Ph.D.; and Christian Grattan, Ph.D.

Location

Richardson Ballroom – DiGiorgio Campus Center

Start Date

12-4-2019 2:15 PM

End Date

April 2019

Description

Xanthomonas cynarae is a phytopathogenic bacterium responsible for causing blight on artichokes, resulting in crop loss. The presence of the bacterium on artichoke leaf tissue triggers a hypersensitive response, a type of programmed cell death characterized by tissue collapse, electrolyte leakage, and extensive modifications to the cell walls and surrounding apoplastic tissues. The hypersensitive response is triggered by the interaction of bacterial avirulence proteins (Avr proteins) with plant resistance proteins (R proteins) in the plant cytosol. The Avr proteins are injected into the plant cell via the Type III Secretion System, a modified flagellum that serves as a molecular needle, unfolding the proteins on the bacterial end and injecting them into the plant cytosol. We have recently identified a protein, named XopAZ, that may aid in the proper refolding of these injected Avr proteins. This protein has shown sequence identity to the FK506 binding protein family of peptidyl-prolyl isomerases and the Sensitive to lysis (Slp) family of chaperonins. We have cloned the gene encoding XopAZ from X. cynarae into a prokaryotic expression plasmid and purified recombinant XopAZ from the resulting bacterial culture. The recombinant protein was purified using metal-chelating affinity and gel filtration chromatographic methods. The resulting purified protein was screened to find conditions suitable for crystal growth in attempts at structural characterization of the protein.

Previously Presented/Performed?

Southeast Regional Meeting of the American Chemical Society (SERMACS), Augusta, Georgia, November 2018

Grant Support?

Supported by an SC INBRE grant from the National Institute for General Medical Sciences (NIH-NIGMS)

Course Assignment

CHEM 551, 552H – Hanna

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Apr 12th, 2:15 PM Apr 12th, 4:15 PM

Expression, Purification and Preliminary Crystallization of a Putative Chaperonin from Xanthomonas cynarae

Richardson Ballroom – DiGiorgio Campus Center

Xanthomonas cynarae is a phytopathogenic bacterium responsible for causing blight on artichokes, resulting in crop loss. The presence of the bacterium on artichoke leaf tissue triggers a hypersensitive response, a type of programmed cell death characterized by tissue collapse, electrolyte leakage, and extensive modifications to the cell walls and surrounding apoplastic tissues. The hypersensitive response is triggered by the interaction of bacterial avirulence proteins (Avr proteins) with plant resistance proteins (R proteins) in the plant cytosol. The Avr proteins are injected into the plant cell via the Type III Secretion System, a modified flagellum that serves as a molecular needle, unfolding the proteins on the bacterial end and injecting them into the plant cytosol. We have recently identified a protein, named XopAZ, that may aid in the proper refolding of these injected Avr proteins. This protein has shown sequence identity to the FK506 binding protein family of peptidyl-prolyl isomerases and the Sensitive to lysis (Slp) family of chaperonins. We have cloned the gene encoding XopAZ from X. cynarae into a prokaryotic expression plasmid and purified recombinant XopAZ from the resulting bacterial culture. The recombinant protein was purified using metal-chelating affinity and gel filtration chromatographic methods. The resulting purified protein was screened to find conditions suitable for crystal growth in attempts at structural characterization of the protein.