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

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 osm...

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Autores principales: Hofmann, Alexander, Müller, Sophia, Drechsler, Thomas, Berleth, Mareike, Caesar, Katharina, Rohr, Leander, Harter, Klaus, Groth, Georg
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154865/
https://www.ncbi.nlm.nih.gov/pubmed/32121559
http://dx.doi.org/10.3390/plants9030304
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author Hofmann, Alexander
Müller, Sophia
Drechsler, Thomas
Berleth, Mareike
Caesar, Katharina
Rohr, Leander
Harter, Klaus
Groth, Georg
author_facet Hofmann, Alexander
Müller, Sophia
Drechsler, Thomas
Berleth, Mareike
Caesar, Katharina
Rohr, Leander
Harter, Klaus
Groth, Georg
author_sort Hofmann, Alexander
collection PubMed
description 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.
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spelling pubmed-71548652020-04-21 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 Plants (Basel) Article 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. MDPI 2020-03-01 /pmc/articles/PMC7154865/ /pubmed/32121559 http://dx.doi.org/10.3390/plants9030304 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hofmann, Alexander
Müller, Sophia
Drechsler, Thomas
Berleth, Mareike
Caesar, Katharina
Rohr, Leander
Harter, Klaus
Groth, Georg
High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title_full High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title_fullStr High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title_full_unstemmed High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title_short High-Level Expression, Purification and Initial Characterization of Recombinant Arabidopsis Histidine Kinase AHK1
title_sort high-level expression, purification and initial characterization of recombinant arabidopsis histidine kinase ahk1
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154865/
https://www.ncbi.nlm.nih.gov/pubmed/32121559
http://dx.doi.org/10.3390/plants9030304
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