Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria.
The isolation and characterization of a monoclonal antibody (MAb 2G5) specific for the bacterial DnaK (HSP70) protein is described. The 2G5 MAb was initially selected because of its ability to bind to DnaK under denaturing conditions. Isotype analyses indicated that 2G5 was an immunoglobulin G2a. Dose-response Western blot (immunoblot) experiments with purified but unconcentrated 2G5 permitted detection of 10 ng of pure DnaK protein. The DnaK epitope was determined by Western blot analysis of a series of truncated DnaK fragments overproduced in Escherichia coli using 5' and 3' dnaK-deleted expression plasmids. The epitope mapped to a 22-amino-acid region spanning DnaK residues 288 and 310. Phylogenetic distribution of the epitope was examined by Western blot analysis of a wide variety of bacterial species and indicated that the epitope was uniquely present in gram-negative organisms. The proximity of the epitope to the presumed DnaK ATP-binding pocket suggested that MAb binding might inhibit DnaK ATPase activity. In vitro analysis supported this prediction and demonstrated that MAb-mediated inhibition of ATPase activity was antibody specific and occurred at stoichiometric molar ratios of MAb to DnaK. Possible mechanisms to explain the ability of the 2G5 MAb to inhibit DnaK activity are discussed.
Krska
J
J
School of Biological Sciences, University of Nebraska, Lincoln 68588-0118.
Elthon
T
T
Blum
P
P
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology; D015180:Electrophoresis, Gel, Two-Dimensional; D000939:Epitopes; D029968:Escherichia coli Proteins; D006090:Gram-Negative Bacteria / Q000276:immunology*; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D007132:Immunoglobulin Isotypes / Q000276:immunology; D010802:Phylogeny; D011994:Recombinant Proteins / Q000276:immunology; D017384:Sequence Deletion; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D007132:Immunoglobulin Isotypes; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C063842:dnaK protein, E coli