Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein

J. Partridge, J. King, J. Krska, D. Rockabrand, Paul H Blum

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

The dnaK (hsp70) gene from the facultative intracellular pathogen Erysipelothrix rhusiopathiae was cloned by heterologous DNA hybridization of a genomic library using the Escherichia coli dnaK gene as a probe. A 3.2-kb fragment which encoded an 1,800-bp open reading frame was recovered. The deduced amino acid sequence of this open reading frame shares 56% identity with the E. coli DnaK protein. Expression of the encoded protein in E. coli by using the phage T7 promoter/polymerase system resulted in accumulation of a unique 65-kDa protein. Western blot (immunoblot) analysis of extracts from a recombinant E. coli strain using anti-E. coli DnaK polyclonal antibodies confirmed that the cloned gene encodes a DnaK homolog. The recombinant E. rhusiopathiae DnaK protein was purified to 80% homogeneity by ATP affinity chromatography. The purified material hydrolyzed ATP with a specific activity of 100 nmol min-1 mg of protein-1. Analysis of total protein extracts from E. rhusiopathiae indicates that DnaK is a highly expressed protein in this organism.

Original languageEnglish (US)
Pages (from-to)411-417
Number of pages7
JournalInfection and Immunity
Volume61
Issue number2
StatePublished - Feb 2 1993

Fingerprint

Erysipelothrix
Organism Cloning
Escherichia coli
Proteins
Open Reading Frames
Adenosine Triphosphate
Western Blotting
Genes
Bacteriophage T7
Genomic Library
Escherichia coli Proteins
Affinity Chromatography
Amino Acid Sequence
Antibodies
DNA

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

Cite this

Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein. / Partridge, J.; King, J.; Krska, J.; Rockabrand, D.; Blum, Paul H.

In: Infection and Immunity, Vol. 61, No. 2, 02.02.1993, p. 411-417.

Research output: Contribution to journalArticle

Partridge, J. ; King, J. ; Krska, J. ; Rockabrand, D. ; Blum, Paul H. / Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein. In: Infection and Immunity. 1993 ; Vol. 61, No. 2. pp. 411-417.
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