Expression and Purification of AvrBs1.1, a Protein Tyrosine Phosphatase from Xanthomonas euvesicatoria
Poster Number
043
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
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.
Honors Thesis Committee
Jason Hurlbert, Ph.D.; Fatima Amir, Ph.D.; and Clifton Harris, Ph.D.
Course Assignment
CHEM 551, 552 – Hanna
Start Date
12-4-2019 2:15 PM
End Date
April 2019
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.
