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

J. Krska, Thomas E Elthon, Paul H Blum

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 G(2a). 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.

Original languageEnglish (US)
Pages (from-to)6433-6440
Number of pages8
JournalJournal of bacteriology
Volume175
Issue number20
DOIs
StatePublished - Jan 1 1993

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Gram-Negative Bacteria
Epitopes
Monoclonal Antibodies
Western Blotting
Adenosine Triphosphatases
Proteins
Plasmids
Immunoglobulin G
Adenosine Triphosphate
Escherichia coli
Amino Acids
Antibodies

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

Cite this

Monoclonal antibody recognition and function of a DnaK (HSP70) epitope found in gram-negative bacteria. / Krska, J.; Elthon, Thomas E; Blum, Paul H.

In: Journal of bacteriology, Vol. 175, No. 20, 01.01.1993, p. 6433-6440.

Research output: Contribution to journalArticle

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