Event Title

Expression and Purification of AvrBs1.1, a Protein Tyrosine Phosphatase from Xanthomonas euvesicatoria

Poster Number

043

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 Clifton Harris, Ph.D.

Location

Richardson Ballroom – DiGiorgio Campus Center

Start Date

12-4-2019 2:15 PM

End Date

April 2019

Description

Bacterial spot of pepper and tomato is a plant disease that leads to significant crop loss from the shedding of pepper and tomato blossoms and young, developing fruit. Xanthomonas euvesicatoria is a phytopathogen that, when infecting plants, causes bacterial spot. Genomic studies of these strains of Xanthomonas have revealed several conserved genes that encode effector proteins that contribute to the virulence of the bacteria. One of these effector proteins, AvrBs1.1, has been identified as a phosphatase by bioinformatic studies. AvrBs1.1 elicits a hypersensitive response (HR) in plants when injected by the Type III Secretion System. The goal of this research was to successfully express and purify AvrBs1.1. The AvrBs1.1::pET28a plasmid DNA was used to transform E.coli BL21(DE3) cultures, which were then used to produce the recombinant AvrBs1.1 protein. Cell pellets of E.coli BL21(DE3) AvrBs1.1::pET28a were suspended in 50 mM TrisHCl, 250 mM NaCl, pH 7.2, and lysed via sonication. The recombinant protein in the supernatant was then purified via metal-chelating affinity chromatography with a Ni2+ HisTrap FF column. Analysis of the column eluate revealed contaminating proteins, indicating that AvrBs1.1 was not completely purified. Other chromatographic methods, such as anion-exchange chromatography, may be more successful in the future. Growing the cells in a different E.coli strain or at a different temperature may also yield more pure protein.

Course Assignment

CHEM 551, 552 – Hanna

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

Expression and Purification of AvrBs1.1, a Protein Tyrosine Phosphatase from Xanthomonas euvesicatoria

Richardson Ballroom – DiGiorgio Campus Center

Bacterial spot of pepper and tomato is a plant disease that leads to significant crop loss from the shedding of pepper and tomato blossoms and young, developing fruit. Xanthomonas euvesicatoria is a phytopathogen that, when infecting plants, causes bacterial spot. Genomic studies of these strains of Xanthomonas have revealed several conserved genes that encode effector proteins that contribute to the virulence of the bacteria. One of these effector proteins, AvrBs1.1, has been identified as a phosphatase by bioinformatic studies. AvrBs1.1 elicits a hypersensitive response (HR) in plants when injected by the Type III Secretion System. The goal of this research was to successfully express and purify AvrBs1.1. The AvrBs1.1::pET28a plasmid DNA was used to transform E.coli BL21(DE3) cultures, which were then used to produce the recombinant AvrBs1.1 protein. Cell pellets of E.coli BL21(DE3) AvrBs1.1::pET28a were suspended in 50 mM TrisHCl, 250 mM NaCl, pH 7.2, and lysed via sonication. The recombinant protein in the supernatant was then purified via metal-chelating affinity chromatography with a Ni2+ HisTrap FF column. Analysis of the column eluate revealed contaminating proteins, indicating that AvrBs1.1 was not completely purified. Other chromatographic methods, such as anion-exchange chromatography, may be more successful in the future. Growing the cells in a different E.coli strain or at a different temperature may also yield more pure protein.