蛋白激酶BIN2的纯化及活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2016-1167

生物技术通报 ›› 2017, Vol. 33 ›› Issue (7): 145-149.doi: 10.13560/j.cnki.biotech.bull.1985.2016-1167

蛋白激酶BIN2的纯化及活性分析

袁敏¹,齐玉荣²,王瑞菊²,

1. 华北理工大学生命科学学院基因组学与计算生物学研究中心，唐山 063000；
2. 河北师范大学生命科学学院，石家庄 050024

收稿日期:2016-12-26 出版日期:2017-07-11 发布日期:2017-07-11
作者简介:袁敏，女，博士，研究方向：植物生长发育；E-mail：yuanmin308@163.com
基金资助:
国家自然科学基金项目（31401212，31201063），河北省自然科学基金（C2014209134，C2012205042）

Purification and Activity Analysis of Tag-free Protein Kinase BIN2

YUAN Min¹, QI Yu-rong² ,WANG Rui-ju²

1. Center for Genomics and Computational Biology，College of Life Sciences，North China University of Science and Technology，Tangshan 063000；
2. College of Life Sciences，Hebei Normal University，Shijiazhuang 050024

Received:2016-12-26 Published:2017-07-11 Online:2017-07-11

摘要/Abstract

摘要： BIN2是一种丝氨酸/苏氨酸蛋白激酶，在油菜素内酯（BR）信号转导中发挥重要作用。为了获得具有激酶活性且不带标签的BIN2蛋白，将BIN2基因构建到带eXact标签的pPAL8表达载体上，获得BIN2基因的原核表达载体eXact-BIN2，转化大肠杆菌表达菌株BL21（DE3）。摸索合适的诱导条件后成功诱导出51 kD 的eXact-BIN2蛋白。经eXact纯化介质纯化并切除标签，获得质量较高、不带标签的BIN2蛋白。该BIN2蛋白具有较高的激酶活性，能够在体外磷酸化MBP-BZR1蛋白。

关键词: BIN2, 磷酸化, 蛋白激酶, 蛋白纯化

Abstract: BIN2 is a kind of Ser/Thr protein kinase，and plays important roles in BR signal transduction pathway. In order to obtain the active and tag-free BIN2 protein，the BIN2 gene was cloned into the prokaryotic expression vector pPAL8 to obtain BIN2-pPAL8 expression vector. The correct plasmid BIN2-pPAL8 was transformed into E.coli expression strain BL21（DE3）. After exploring the proper induction conditions，the 51 kD eXact-BIN2 protein was successfully induced and purified using eXact purification system. The high-quality and tag-free BIN2 protein was obtained through cleaving the eXact tag. The purified BIN2 protein is highly active，which could phosphorylate MBP-BZR1 in vitro.

Key words: BIN2, phosphorylation, protein kinase, protein purification

袁敏,齐玉荣,王瑞菊,. 蛋白激酶BIN2的纯化及活性分析[J]. 生物技术通报, 2017, 33(7): 145-149.

YUAN Min, QI Yu-rong ,WANG Rui-ju. Purification and Activity Analysis of Tag-free Protein Kinase BIN2[J]. Biotechnology Bulletin, 2017, 33(7): 145-149.

参考文献

[1] Li J, Nam KH. Regulation of brassinosteroid signaling by a GSK3/SHAGGY-like kinase[J] . Science, 2002, 295（5558）：1299-1301.
[2] Yan ZY, Zhao J, Peng P, et al. BIN2 functions redundantly with other Arabidopsis GSK3-Like kinases to regulate brassinosteroid signaling[J] . Plant Physiology, 2009, 150（2）：710-721.
[3] Kim TW, Guan S, Sun Y, et al. Brassinosteroid signal transduction from cell-surface receptor kinases to nuclear transcription factors[J] . Nature Cell Biology, 2009, 11（10）：1254-1260.
[4] Kim TW, Michniewicz M, Bergmann DC, et al. Brassinosteroid regulates stomatal development by GSK3-mediated inhibition of a MAPK pathway[J] . Nature, 2012, 482（7385）：419-U1526.
[5] Khan M, Rozhon W, Bigeard J, et al. Brassinosteroid-regulated GSK3/Shaggy-like kinases phosphorylate mitogen-activated protein（MAP）kinase kinases, which control stomata development in Arabidopsis thaliana[J] . Journal of Biological Chemistry, 2013, 288（11）：7519-7527.
[6] Cho H, Ryu H, Rho S, et al. A secreted peptide acts on BIN2-mediated phosphorylation of ARFs to potentiate auxin response during lateral root development[J] . Nature Cell Biology, 2014, 16（1）：66-76.
[7] Vert G, Walcher CL, Chory J, et al. Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2[J] . Proceedings of the National Academy of Sciences of the United States of America, 2008, 105（28）：9829-9834.
[8] Dal Santo S, Stampfl H, Krasensky J, et al. Stress-induced GSK3 regulates the redox stress response by phosphorylating glucose-6-phosphate dehydrogenase in Arabidopsis[J] . Plant Cell, 2012, 24（8）：3380-3392.
[9] Wang RJ, Liu MM, Yuan M, et al. The Brassinosteroid-activated BRI1 receptor kinase is switched off by dephosphorylation mediated by cytoplasm-localized PP2A B' Subunits[J] . Molecular Plant, 2016, 9（1）：148-157.
[10] Clouse SD, Langford M, McMorris TC. A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development[J] . Plant Physiology, 1996, 111（3）：671-678.
[11] Li J, Nagpal P, Vitart V, et al. A role for brassinosteroids in light-dependent development of Arabidopsis[J] . Science, 1996, 272（5260）：398-401.
[12] Kim TW, Wang ZY. Brassinosteroid signal transduction from receptor kinases to transcription factors[J] . Annual Review of Plant Biology, 2010, 61：681-704.
[13] Tang W, Yuan M, Wang R, et al. PP2A activates brassinosteroid-responsive gene expression and plant growth by dephosphorylating BZR1[J] . Nature Cell Biology, 2011, 13（2）：124-131.
[14] Kim TW, Guan S, Burlingame AL, et al. The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2[J] . Molecular Cell, 2011, 43（4）：561-571.
[15] Hartwig T, Wang ZY. The molecular circuit of steroid signalling in plants[J] . Essays Biochem 2015, 58：71-82.
[16] Fan M, Bai MY, Kim JG, et al. The bHLH transcription factor HBI1 mediates the trade-off between growth and pathogen-associated molecular pattern-triggered immunity in Arabidopsis[J] . Plant Cell, 2014, 26（2）：828-841.
[17] Gudesblat GE, Schneider-Pizon J, Betti C, et al. SPEECHLESS integrates brassinosteroid and stomata signalling pathways[J] . Nature Cell Biology, 2012, 14（5）：548-U214.
[18] Huang L, Mao X, Abdulaev NG, et al. One-step purification of a functional, constitutively activated form of visual arrestin[J] . Protein Expression And Purification, 2012, 82（1）：55-60.
[19] 李青, 张强, 邹丽云, 等. 应用 Profinity eXact 标签在真核表达系统中纯化 EGFP 蛋白[J] . 免疫学杂志, 2011, 27（2）：144-148.
[20] 冯金, 洪愉, 黄芬, 等. 基于 Profinity eXact 系统的可溶性 egfp 表达和纯化[J] . 生命科学研究, 2014, 18（4）：310-314.

蛋白激酶BIN2的纯化及活性分析

Purification and Activity Analysis of Tag-free Protein Kinase BIN2

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	杨玉梅, 张坤晓. 应用CRISPR/Cas9技术建立ERK激酶相分离荧光探针定点整合的稳定细胞株[J]. 生物技术通报, 2023, 39(8): 159-164.
[2]	刘铖霞, 孙宗艳, 罗云波, 朱鸿亮, 曲桂芹. bHLH转录因子的磷酸化调控植物生理功能的研究进展[J]. 生物技术通报, 2023, 39(3): 26-34.
[3]	覃雪晶, 王雨涵, 曹一博, 张凌云. 青杄PwHAP5基因原核表达及多克隆抗体制备[J]. 生物技术通报, 2022, 38(8): 142-149.
[4]	梁星星, 王佳, 许文涛. 抗病毒核苷酸类似物磷酸化修饰研究进展[J]. 生物技术通报, 2022, 38(2): 218-226.
[5]	孙忠娟, 刘倩倩, 郭雨纤, 王光辉, 王晨芳. Analog-sensitive蛋白激酶研究系统在植物病原真菌中的建立[J]. 生物技术通报, 2022, 38(11): 49-57.
[6]	刘媛媛, 杨冬杰, 左东云, 程海亮, 张友平, 吕丽敏, 王巧连, 宋国立. 棉花GhD6PKL2的克隆及功能验证[J]. 生物技术通报, 2021, 37(8): 111-120.
[7]	段绪果, 张玉华, 黄婷婷, 丁乾, 栾舒越, 朱秋雨. 化学分子伴侣及诱导条件协同强化Thermotoga maritima α-葡聚糖磷酸化酶可溶性表达[J]. 生物技术通报, 2021, 37(8): 233-242.
[8]	唐禄, 董丽平, 尹茉莉, 刘磊, 董媛, 王会岩. 成纤维细胞生长因子20单克隆抗体的制备及鉴定[J]. 生物技术通报, 2021, 37(10): 179-185.
[9]	刘静, 李亚超, 周梦岩, 吴鹏飞, 马祥庆, 李明. 植物蛋白质翻译后修饰组学研究进展[J]. 生物技术通报, 2021, 37(1): 67-76.
[10]	孟利, 杜彩萍. 大鼠His-Akt1重组蛋白的真核表达、蛋白纯化及活性鉴定[J]. 生物技术通报, 2020, 36(12): 98-103.
[11]	周阳, 王桃桃, 闫丹丹, 王莹莹, 施沁璇, 孙丽慧, 林锋. 类弹性蛋白作为功能纳米材料在生物工程领域研究及应用进展[J]. 生物技术通报, 2020, 36(11): 198-208.
[12]	侯成林, 杨艳坤, 陈嘉荔, 白仲虎. Mxr1磷酸化水平受Ptp调控机理的初步研究[J]. 生物技术通报, 2019, 35(7): 108-113.
[13]	王海波, 郭俊云, 田雪莲. 小桐子SnRK1蛋白激酶α亚基基因的克隆及原核表达分析[J]. 生物技术通报, 2019, 35(6): 39-47.
[14]	周利明, 卢鑫蕊, 马声葳, 房玮. 钙依赖蛋白激酶CPK14在花粉管生长中的功能分析[J]. 生物技术通报, 2019, 35(6): 55-61.
[15]	李玲, 白娟娟, 郭梅, 王思禹, 王晓玥. 番茄果实转录因子CNR的原核表达与纯化及其抗体的制备[J]. 生物技术通报, 2019, 35(2): 1-8.