Schematic diagram of VapC purification, sequence-specificity, and kinetic analysis. To neutralize VapC toxicity it is expressed in complex with VapB. Proteolyticly sensitive VapB is removed by trypsin, and VapC is purified by anion exchange chromatography. RNase activity and sequence-specificity are screened using Pentaprobes (ticks indicate Pentaprobes cut by all VapC proteins tested; crosses indicate Pentaprobes not cut). Short RNA oligonucleotides are designed based on the Pentaprobe RNA cleaved by all VapC proteins and cut sites determined by MALDI-TOF MS and in-house software. A fluorogenic RNA substrate with a single cut site is then designed for determination of VapC kinetic parameters.
