PURIFICATION OF 2 BACILLUS-SUBTILIS PROTEINS WHICH CROSS-REACT WITH ANTIBODIES DIRECTED AGAINST EUKARYOTIC PROTEIN-KINASE-C, THE HIS HPR KINASE AND TRIGGER FACTOR

As in eukaryotes, phosphorylation of Ser and Thr residues in proteins appears to be a common phenomenon in bacteria. Surprisingly, however, very few Ser/Thr protein kinases have been identified and in this stud y antibodies directed against mammalian protein kinase C (PKC) have be en used in attempts to isolate conserved Ser/Thr protein kinases. Usin g the mAb M7 against rat brain PKC, a single 70 kDa band was identifie d in total cell extracts of Bacillus subtilis by Western blotting afte r SDS-PACE, whilst using polyclonal antibody alpha-PKC1p against Sacch aromyces cerevisiae PKC a single 67 kDa band was identified by the sam e procedure. The two proteins were purified independently on the basis of antibody recognition employing two-dimensional gel electrophoresis as a final step, which allowed subsequent microsequencing. The 70 kDa band was thus identified as the phosphoenolpyruvate-dependent His HPr kinase, Enzyme I of the phosphotransferase system. This identity was confirmed using a mutant deleted for ptsl, encoding Enzyme I. The 67 k Da protein was identified as a previously unknown B. subtilis 'trigger factor', homologous to an Escherichia coli protein-folding enzyme, pe ptidylprolyl cis-trans-isomerase implicated in cell division.