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<b>Abstract</b>— <p>In this study, we investigated thermosensitive histidine kinase DesK from <i>Bacillus subtilis</i> 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.</p>

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

  • 期刊论文

  • 发表日期：

    2025年3月1日

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  • D. D. Kuklina,
  • T. N. Murugova,
  • M. I. Rulev,
  • Yu. L. Ryzhykau

摘要

Abstract—

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.