A general procedure for the isolation of heat-shock proteins from periodontopathogenic bacteria

The isolation of heat-shock proteins (HSP) from whole cells of Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans and Bacteroides forsythus, which are recognized as periodontopathogens, was performed by affinity chromatography on adenosine 5'-triphosphate-agarose followed by preparative polyacrylamide gel electrophoresis under denaturing conditions. Analysis of the final material by silver nitrate staining of SDS-PAGE gels and Western immunoblotting using a commercial polyclonal antibody against HSP60 from Synechococcus sp. revealed homogenous protein preparations corresponding to GroEL-like proteins with apparent molecular mass of 68 kDa (P. gingivalis), 64 kDa (A. actinomycetemcomitans) and 67 kDa (B. forsythus). A second HSP was also isolated from P. gingivalis and B. forsythus, and recognized as a DnaK-like protein by immunoblotting with polyclonal antibody against DnaK (HSP70) from Escherichia coli. Our HSP isolation procedure is rapid and simple, and the purified proteins obtained may be used to determine their homology with other HSP previously characterized and to raise antibodies.