Expression, Purification and Preliminary Crystallization of a Putative Chaperonin from Xanthomonas cynarae
Poster Number
037
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
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.
Honors Thesis Committee
Jason Hurlbert, Ph.D.; Fatima Amir, Ph.D.; and Christian Grattan, Ph.D.
Course Assignment
CHEM 551, 552H – Hanna
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)
Start Date
12-4-2019 2:15 PM
End Date
April 2019
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.
