High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

Hofmann, Alexander; Müller, Sophia; Drechsler, Thomas; Berleth, Mareike; Caesar, Katharina; Rohr, Leander; Harter, Klaus; Groth, Georg

doi:10.3390/plants9030304

Open AccessArticle

High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

by

Alexander Hofmann

^1,†

,

Sophia Müller

^1,†,

Thomas Drechsler

^2,†

,

Mareike Berleth

¹,

Katharina Caesar

²,

Leander Rohr

²

,

Klaus Harter

²

and

Georg Groth

^1,*

¹

Institute of Biochemical Plant Physiology, Heinrich Heine University, Düsseldorf 40225, Germany

²

Center for Plant Molecular Biology (ZMBP), Eberhard Karls Universität Tübingen, Tübingen 72076, Germany

^*

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Plants 2020, 9(3), 304; https://doi.org/10.3390/plants9030304

Submission received: 14 January 2020 / Revised: 18 February 2020 / Accepted: 24 February 2020 / Published: 1 March 2020

(This article belongs to the Special Issue The Plant Two-Component System)

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Abstract

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.

Keywords: histidine kinase; osmosensing; membrane protein purification; phosphorelay; protein-protein interaction

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MDPI and ACS Style

Hofmann, A.; Müller, S.; Drechsler, T.; Berleth, M.; Caesar, K.; Rohr, L.; Harter, K.; Groth, G. High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1. Plants 2020, 9, 304. https://doi.org/10.3390/plants9030304

AMA Style

Hofmann A, Müller S, Drechsler T, Berleth M, Caesar K, Rohr L, Harter K, Groth G. High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1. Plants. 2020; 9(3):304. https://doi.org/10.3390/plants9030304

Chicago/Turabian Style

Hofmann, Alexander, Sophia Müller, Thomas Drechsler, Mareike Berleth, Katharina Caesar, Leander Rohr, Klaus Harter, and Georg Groth. 2020. "High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1" Plants 9, no. 3: 304. https://doi.org/10.3390/plants9030304

APA Style

Hofmann, A., Müller, S., Drechsler, T., Berleth, M., Caesar, K., Rohr, L., Harter, K., & Groth, G. (2020). High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1. Plants, 9(3), 304. https://doi.org/10.3390/plants9030304

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High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1

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