Co‐expression and purification of a toxin‐antitoxin VapC–VapB complex from the E. coli O145:H28 strain RM12581 associated with a Romain lettuce outbreak.

Abstract

VapC and VapB are the toxin and anti‐toxin proteins, respectively, of the VapCB toxin–antitoxin system found in bacteria and associated with stunting cell growth during stressful conditions. VapC homologs typically are tRNA endonucleases, and VapB homologs are known to bind to double‐stranded DNA, as well as VapC to inihibit its endonuclease acitivty. No cognate binding and/or substrate partners have been identified for E. coli strain here VapC or VapB homologs. Our goal is to determine the X‐ray crystal structure of an E. coli O145:H28 strain RM12581 VapC complexed with antitoxin VapB or to any tRNA substrate that we are able to identify. I will present our efforts to produce soluble relevant proteins and/or complex thereof. A DNA co‐expression vector encoding both VapB and VapC mimicking its natural expression context was transformed into E. coli BL21 (DE3) competent cells and protein expression was induced. Proteins were purified using nickel affinity chromatography and size exclusion chromatography. Currently crystal screening is underway to crystallize relevant proteins for subsequent X‐ray diffraction analysis.