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

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.

Mechanisms of plant cell wall surveillance in response to pathogens, cell wall-derived ligands and the effect of expansins to infection resistance or susceptibility

“…The best characterized osmo-receptors in plants are the Arabidopsis thaliana histidine protein kinases (HPKs) ( Pham et al, 2012 ). The A. thaliana genome contains eleven HPKs, of which five have been recognized as ethylene receptors, three as cytokinin receptors and the remaining three have no known activity as hormone receptors, although they participate in other signaling processes including responses to biotic and abiotic stresses ( Pham et al, 2012 ; Hofmann et al, 2020 ). An additional mechanism to cope with physical forces is mediated by mechano-sensitive ion channels (MSIC) and mechano-sensitive like channels (MSL) located at the inner chloroplast envelope ( Haswell and Meyerowitz, 2006 ; Bacete and Hamann, 2020 ).…”

Novel foam separation column with hollow octagonal prismoids with sieve tray for purification of recombinant β-glucosidase

“…Fusion proteins constructed by fusion tag technology have been successfully expressed in numerous microbial host cells, and multifunctional recombinant proteins have positive signi cance in improving protein expression and simplifying protein puri cation [24,25]. At present, commonly used fusion tags are including glutathione S-transferase (GST) [26], streptavidin binding peptide (SBP) [27], and histidine (His) [28], etc.…”

The unknown soldier in citrus plants: polyamines-based defensive mechanisms against biotic and abiotic stresses and their relationship with other stress-associated metabolites

“…The putrescine, spermidine, and spermine are the major ubiquitously found PAs in higher plants. [22][23][24] In citrus, PAs are synthesized from L -arginine through two different routes (well-reviewed recently by Killiny and Nehela, 2020). 25 The first route is where L -arginine is converted by arginaseto ornithine, which is then decarboxylated by ornithine decarboxylase (ODC) to form the diamine putrescine.…”

“…[22][23][24] In citrus, PAs are synthesized from L -arginine through two different routes (well-reviewed recently by Killiny and Nehela, 2020). 25 The first route is where L -arginine is converted by arginaseto ornithine, which is then decarboxylated by ornithine decarboxylase (ODC) to form the diamine putrescine. The second route involving arginine decarboxylation by arginine decarboxylase (ADC) to produce agmatine, which catalyzes toputrescine via some catalytic steps.…”