Monitoring of the heat-shock response in Escherichia coli using an optical biosensor.
A surface plasmon resonance (SPR) method for monitoring the concentration of the chaperone DnaK and its relation to physiological stress response in a recombinant Escherichia coli strain subjected to heat shock is described. The DnaK protein, an abundantly occurring representative of the heat-shock proteins, was used as a marker of physiological stress. The SPR biosensor instrument was used for label-free immunoaffinity detection directly in cell culture lysates using an anti-DnaK monoclonal IgG antibody immobilized on the sensor surface. The SPR method provides a fast response (<8 min) and a reproducible (RSD<2%), accurate (comparison to the direct enzyme-linked immunosorbent assay), and sensitive (LOD<1 nM) assay for determination of the DnaK level in cell culture lysates. The operational stability of the method was high compared to that of other SPR assays; the sensitivity decreased at only 2.7%/h. This allowed measurement of more than 220 samples per sensor surface. Storage stability was determined at 25 degrees C (100% after 17 h) and 10 degrees C (101% after 1 month). The method was validated by standard additions of DnaK (30, 60, and 120 nM) with recovery indices in the range 95.7-103.7%.
Department of Physics and Measurement Technology, Linköping University, 581 83 Linköping, Sweden.
Igor
I
Vostiar
Jan
J
Tkac
Carl Fredrik
CF
Mandenius
D001671:Biochemistry / Q000379:methods; D015374:Biosensing Techniques; D002021:Buffers; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D018869:Heat-Shock Response / Q000502:physiology*; D055095:Optics and Photonics; D015203:Reproducibility of Results; D013048:Specimen Handling / Q000379:methods; D020349:Surface Plasmon Resonance / Q000379:methods*
D002021:Buffers; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; C063842:dnaK protein, E coli