Abstract
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 °C (100% after 17 h) and 10 °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%.
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