Cloning, heterologous expression, and characterization of the Erysipelothrix rhusiopathiae DnaK protein.
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.
Partridge
J
J
School of Biological Science, University of Nebraska, Lincoln 68588-0118.
King
J
J
Krska
J
J
Rockabrand
D
D
Blum
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D004888:Erysipelothrix / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli