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Expression and phosphorylation analysis of soluble proteins and membrane-localised receptor-like kinases from Arabidopsis thaliana in Escherichia coli
J Plant Biotechnol 2018;45:315-321
Published online December 31, 2018
© 2018 The Korean Society for Plant Biotechnology.

Eun-Seok Oh, Foyjunnaher Eva, Sang-Yun Kim, Man-Ho Oh

Department of Biological Sciences, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon 34134, Republic of Korea
Correspondence to: e-mail: manhooh@cnu.ac.kr
Received December 7, 2018; Revised December 13, 2018; Accepted December 13, 2018.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Molecular and functional characterization of proteins and their levels is of great interest in understanding the mechanism of diverse cellular processes. In this study, we report on the convenient Escherichia coli-based protein expression system that allows recombinant of soluble proteins expression and cytosolic domain of membrane-localised kinases, followed by the detection of autophosphorylation activity in protein kinases. This approach is applied to regulatory proteins of Arabidopsis thaliana, including 14-3-3, calmodulin, calcium-dependent protein kinase, TERMINAL FLOWER 1(TFL1), FLOWERING LOCUS T (FT), receptor-like cytoplasmic kinase and cytoplasmic domain of leucine-rich repeat-receptor like kinase proteins. Our Western blot analysis which uses phospho-specific antibodies showed that five putative LRR-RLKs and two putative RLCKs have autophosphorylation activity in vitro on threonine and/or tyrosine residue(s), suggesting their potential role in signal transduction pathways. Our findings were also discussed in the broader context of recombinant expression and biochemical analysis of soluble and membrane-localised receptor kinases in microbial systems.
Keywords : Calcium-dependent protein kinase, Leucine-rich repeat receptor-like kinase (LRR-RLKs), Receptor-like cytoplasmic kinases (RLCKs), Post-translational modification, Arabidopsis thaliana, Escherichia coli


December 2018, 45 (4)
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