Cloning of Streptococcus pyogenes ack and pta Genes, and Purification and Characterization of the Corresponding Recombinant Proteins

Group A Streptococcus (GAS, S. pyogenes) virulence factors are controlled in general by two‐component regulatory systems (TCSs) constituted by a response regulator (RR) and a histidine kinase (HK). RR regulates virulence in GAS and HK phopshorylates it in the presence of ATP. The function of the RR remains unaltered even in the absence of HK. In general, bacteria produce acetyl phosphate through acetate kinase (ACK) and phosphotransacetylase (PTA). It is hypothesized that in the absence of HK, the acetyl phosphate generated from the PTA‐ACK pathway serves as a phosphodonor for the RR. In order to understand the pathway, the pta and ack genes were cloned into pET14B His‐tag vector to make the corresponding recombinant His‐tagged ACK and PTA proteins. These proteins were purified by affinity chromatography using Ni+‐NTA affinity column. The presence of His‐tagged proteins in the crude and purified preparations was confirmed by Western blot technique using anti‐HisX6 polyclonal antibody. Future research on the biochemical functions of these two proteins and the interaction of the energy‐rich acetyl phosphate with various response regulators will provide a better understanding of GAS virulence.