title: In vitro complex-formation between the molecular chaperone DnaK and staphylococcal protein A derivatives produced in Escherichia coli and its use in the purification of DnaK.
abstract: Complex-formation between a truncated staphylococcal Protein A produced in Escherichia coli and a native E coli molecular chaperone, DnaK, can be used for the purification of DnaK by IgG-affinity chromatography. The half-time constant for in vitro formation of the Protein A-DnaK complex is about 14 min. Complex-formation in the presence of ATP is faster, but pre-incubation of DnaK with ATP decreases the final amount of the complex. A second complex with a slower migration on native PAGE is formed when the ratio of DnaK to Protein A is increased. A derivative of Protein A, ZZ, which essentially contains only two modified domains of Protein A, did not bind DnaK. After insertion of a tryptophan-rich peptide close to the C-terminus, the resulting protein, ZZT3, became able to bind DnaK. The binding of these three proteins to DnaK correlates with proteolysis in E coli, indicating a possible role for the binding of DnaK in the control of proteolysis.
authors.affiliation: Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
authors.forename: K
authors.initials: K
authors.lastname: Gustavsson
authors.forename: T
authors.initials: T
authors.lastname: Bergman
authors.forename: A
authors.initials: A
authors.lastname: Veide
authors.forename: S O
authors.initials: SO
authors.lastname: Enfors
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C063842:dnaK protein, E coli