title: Expression, Purification, and Characterization of a Sucrose Nonfermenting 1-Related Protein Kinases 2 ofArabidopsis thalianainE. coli-Based Cell-Free System.
abstract: 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 kinasesin vitrocan 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 modifiedE. colicell-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 improvedE. colicell-free system provides us a promising and convenient platform to enhance expression of the target proteins economically.
authors.affiliation: Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi'an, Shaanxi 710043, China.
authors.forename: Xu
authors.identifier: 0000-0002-8108-3471
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Biomass Energy Center for Arid and Semiarid Lands, College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, China.
authors.forename: Shuangxi
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi'an, Shaanxi 710043, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.affiliation: Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi'an, Shaanxi 710043, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zi
authors.affiliation: Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi'an, Shaanxi 710043, China.
authors.forename: Jianhui
authors.initials: J
authors.lastname: Wang
authors.affiliation: Biomass Energy Center for Arid and Semiarid Lands, College of Life Sciences, Northwest A&F University, 3 Taicheng Road, Yangling, Shaanxi 712100, China.
authors.forename: Shaolin
authors.identifier: 0000-0001-7607-8275
authors.initials: S
authors.lastname: Chen
authors.affiliation: Microbiology Institute of Shaanxi, Shaanxi Academy of Sciences, 76 Xiying Road, Xi'an, Shaanxi 710043, China.
authors.forename: Yi
authors.identifier: 0000-0002-0275-9374
authors.initials: Y
authors.lastname: Wan
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002474:Cell-Free System; D004926:Escherichia coli / Q000737:chemistry*; D015870:Gene Expression; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; C492978:SnRK2 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases