Influence of Expression Temperature on the Adenosine Phosphate Bound State of Recombinant BsDesK

In this study, we investigated thermosensitive histidine kinase DesK from Bacillus subtilis with the aim of obtaining a monodisperse protein preparation suitable for further structural studies. A homogeneous sample, both in terms of oligomeric state and ligand binding, is favorable for electron microscopy or X-ray diffraction applications. The binding of ATP is required for autophosphorylation activity of DesK, thus we aimed at developing heterologous expression techniques that would yield ATP-bound protein. We focused on two critical factors that may influence ATP binding: the nutrient composition of the growth medium and the expression temperature. Protein characterization was performed using size-exclusion chromatography, with absorbance monitored at both 280 and 260 nm to assess protein homogeneity and nucleotide binding. Our results indicate that while nutrient composition had a negligible effect on the relative amount of ATP-bound protein, expression temperature played a significant role. Specifically, lowering the expression temperature from 37°C to 20°C at the time of induction markedly increased the proportion of ATP-bound DesK oligomers.