Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System
The plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation...
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doaj-54a4e9f3bea34f4b8dcd98be090c72622020-11-24T23:10:43ZengHindawi LimitedBioMed Research International2314-61332314-61412016-01-01201610.1155/2016/94693569469356Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free SystemXu Zhang0Shuangxi Zhang1Jun Wang2Jing Zi3Jianhui Wang4Shaolin Chen5Yi Wan6Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi’an, Shaanxi 710043, ChinaBiomass Energy Center for Arid and Semiarid Lands, College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, ChinaMicrobiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi’an, Shaanxi 710043, ChinaMicrobiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi’an, Shaanxi 710043, ChinaMicrobiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi’an, Shaanxi 710043, ChinaBiomass Energy Center for Arid and Semiarid Lands, College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, ChinaMicrobiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi’an, Shaanxi 710043, ChinaThe plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation responses in Arabidopsis. Large-scale production of SnRK2 kinases in vitro can help to elucidate the biochemical properties and physiological functions of this protein family. However, heterogenous expression of SnRK2s usually leads to inactive proteins. In this study, we expressed a recombinant Arabidopsis SnRK2.1 in a modified E. coli cell-free system, which combined two kinds of extracts allowing for a convenient and affordable protein preparation. The recombinant SnRK2.1 was produced in large-scale and the autophosphorylation activity of purified SnRK2.1 was characterized, allowing for further biochemical and substrate binding analysis in sulfur signaling. The application of this improved E. coli cell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically.http://dx.doi.org/10.1155/2016/9469356 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Xu Zhang Shuangxi Zhang Jun Wang Jing Zi Jianhui Wang Shaolin Chen Yi Wan |
spellingShingle |
Xu Zhang Shuangxi Zhang Jun Wang Jing Zi Jianhui Wang Shaolin Chen Yi Wan Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System BioMed Research International |
author_facet |
Xu Zhang Shuangxi Zhang Jun Wang Jing Zi Jianhui Wang Shaolin Chen Yi Wan |
author_sort |
Xu Zhang |
title |
Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System |
title_short |
Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System |
title_full |
Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System |
title_fullStr |
Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System |
title_full_unstemmed |
Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 of Arabidopsis thaliana in E. coli-Based Cell-Free System |
title_sort |
expression, purification, and characterization of a sucrose nonfermenting 1-related protein kinases 2 of arabidopsis thaliana in e. coli-based cell-free system |
publisher |
Hindawi Limited |
series |
BioMed Research International |
issn |
2314-6133 2314-6141 |
publishDate |
2016-01-01 |
description |
The plant-specific sucrose nonfermenting 1-related protein kinase 2 (SnRK2) family is considered an important regulator of plant responses to abiotic stresses such as drought, cold, salinity, and nutrition deficiency. However, little information is available on how SnRK2s regulate sulfur deprivation responses in Arabidopsis. Large-scale production of SnRK2 kinases in vitro can help to elucidate the biochemical properties and physiological functions of this protein family. However, heterogenous expression of SnRK2s usually leads to inactive proteins. In this study, we expressed a recombinant Arabidopsis SnRK2.1 in a modified E. coli cell-free system, which combined two kinds of extracts allowing for a convenient and affordable protein preparation. The recombinant SnRK2.1 was produced in large-scale and the autophosphorylation activity of purified SnRK2.1 was characterized, allowing for further biochemical and substrate binding analysis in sulfur signaling. The application of this improved E. coli cell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically. |
url |
http://dx.doi.org/10.1155/2016/9469356 |
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