This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Krska, J Articles by Blum, P Search for Related Content PubMed PubMed Citation Articles by Krska, J Articles by Blum, P

research-article

Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria.

School of Biological Sciences, University of Nebraska, Lincoln 68588-0118.

ABSTRACT

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.

This article has been cited by other articles:

• Ponniah, G., Chen, H., Michielutti, R., Salonen, N., Blum, P. (2003). Single-Cell Protein Profiling of Wastewater Enterobacterial Communities Predicts Disinfection Efficiency. Appl. Environ. Microbiol. 69: 4227-4235 [Abstract] [Full Text]  
• Rockabrand, D., Austin, T., Kaiser, R., Blum, P. (1999). Bacterial Growth State Distinguished by Single-Cell Protein Profiling: Does Chlorination Kill Coliforms in Municipal Effluent?. Appl. Environ. Microbiol. 65: 4181-4188 [Abstract] [Full Text]  
• Weimer, R. M., Creighton, C., Stassinopoulos, A., Youderian, P., Hartzell, P. L. (1998). A Chaperone in the HSP70 Family Controls Production of Extracellular Fibrils in Myxococcus xanthus. J. Bacteriol. 180: 5357-5368 [Abstract] [Full Text]  
• Rolfsmeier, M., Haseltine, C., Bini, E., Clark, A., Blum, P. (1998). Molecular Characterization of the alpha -Glucosidase Gene (malA) from the Hyperthermophilic Archaeon Sulfolobus solfataricus. J. Bacteriol. 180: 1287-1295 [Abstract] [Full Text]  
• Lund, A. A., Blum, P. H., Bhattramakki, D., Elthon, T. E. (1998). Heat-Stress Response of Maize Mitochondria. Plant Physiol. 116: 1097-1110 [Abstract] [Full Text]  
• Rockabrand, D., Livers, K., Austin, T., Kaiser, R., Jensen, D., Burgess, R., Blum, P. (1998). Roles of DnaK and RpoS in Starvation-Induced Thermotolerance of Escherichia coli. J. Bacteriol. 180: 846-854 [Abstract] [Full Text]