Expression and Purification of GeneM: A Novel Virulence Factor of Clavibacter Michiganensis
Session Title
Science and Technology
College
College of Arts and Sciences
Department
Chemistry, Physics, Geology, & the Environment
Abstract
GeneM is a novel virulence factor of unknown structure and function produced by Clavibacter michiganensis, the etiological agent of many diseases in agricultural plants. Tomato and potato plants containing the gene have shown signs of symptomatic necrosis, yet plants containing mutants of the gene were shown to be asymptomatic. BLAST analysis of the amino acid sequence has identified homologous proteins belonging to the patain superfamily, however, bioinformatic analysis of the amino acid sequence and homology modeling contradicts this identification. In this work, we describe our efforts to model the structure of GeneM and express it in recombinant Escherichia coli cultures. Of the three algorithms used to generate a homology model of GeneM, only one gave us a plausible structure. De novo modeling using trRosetta gave a model that is structurally similar to the homology model. Expression trials were performed using different strains of E. coli including BL21, NiCo, and Rosetta 2, and based upon the results, cultures of E. coli BL21 Rosetta 2 (DE3) had a band at the expected size on SDS-PAGE gels. 6L expression cultures were generated and a chromatographic method was developed, involving MCAC, gel-filtration, and cation exchange, to purify the protein for enzymological studies. Future work includes a phospholipase assay.
Start Date
15-4-2022 12:00 PM
Expression and Purification of GeneM: A Novel Virulence Factor of Clavibacter Michiganensis
GeneM is a novel virulence factor of unknown structure and function produced by Clavibacter michiganensis, the etiological agent of many diseases in agricultural plants. Tomato and potato plants containing the gene have shown signs of symptomatic necrosis, yet plants containing mutants of the gene were shown to be asymptomatic. BLAST analysis of the amino acid sequence has identified homologous proteins belonging to the patain superfamily, however, bioinformatic analysis of the amino acid sequence and homology modeling contradicts this identification. In this work, we describe our efforts to model the structure of GeneM and express it in recombinant Escherichia coli cultures. Of the three algorithms used to generate a homology model of GeneM, only one gave us a plausible structure. De novo modeling using trRosetta gave a model that is structurally similar to the homology model. Expression trials were performed using different strains of E. coli including BL21, NiCo, and Rosetta 2, and based upon the results, cultures of E. coli BL21 Rosetta 2 (DE3) had a band at the expected size on SDS-PAGE gels. 6L expression cultures were generated and a chromatographic method was developed, involving MCAC, gel-filtration, and cation exchange, to purify the protein for enzymological studies. Future work includes a phospholipase assay.
