Interaction between heat shock protein DnaK and recombinant staphylococcal protein A.
When a protein derived from the immunoglobulin G (IgG)-binding domains of staphylococcal protein A was expressed in Escherichia coli and recovered from cell extract by IgG affinity chromatography, the 69-kilodalton heat shock protein DnaK was found to be copurified. DnaK could be selectively eluted from the IgG column by ATP or by lowering the pH to 4.7. Protein A could subsequently be eluted by lowering the pH to 3.2. Thus, this procedure allows a one-step purification of both DnaK and protein A from cell extract. In vitro experiments with pure DnaK and protein A revealed that DnaK did not interfere with the IgG-binding properties of protein A but associated with its unfolded C-terminal in a salt-resistant manner. In addition, a specific interaction between DnaK and denaturated casein was found.
Hellebust
H
H
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Uhlén
M
M
Enfors
S O
SO
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000302:isolation & purification / Q000378:metabolism*
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C063842:dnaK protein, E coli