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中国农学通报

中国农学通报 ›› 2019, Vol. 35 ›› Issue (3): 58-61.doi: 10.11924/j.issn.1000-6850.casb18080106

所属专题： 生物技术； 园艺

• 生物技术科学 • 上一篇    下一篇

甜菜M14品系BvM14-STPK蛋白激酶在原核表达系统中低温诱导表达及纯化

于冰, 刘祥, 李海英   

  1. 黑龙江大学 生命科学学院
  • 收稿日期:2018-08-29 修回日期:2018-12-27 接受日期:2018-10-24 出版日期:2019-01-28 发布日期:2019-01-28
  • 通讯作者: 李海英
  • 基金资助:
    国家自然科学基金“甜菜M14 品系抗氧化酶系统响应盐胁迫应答过程的研究”(31471552)；国家自然科学基金“甜菜M14 品系丝氨酸苏氨 酸蛋白激酶基因(BvM14-STPK)响应盐胁迫功能研究”(31501359)；国家自然科学基金“甜菜M14 品系乙二醛酶I 基因抗盐能力的转录因子功能研究” (31671751)；黑龙江省自然科学基金“盐胁迫下甜菜M14 品系根的磷酸化蛋白质组学研究”(C2017057)；黑龙江省高校创新团队建设计划项目“寒区 植物重要基因资源的挖掘与种质创新”(2014TD004)。

Low Temperature Induced Expression and Purification of BvM14-STPK Protein Kinase from Sugar Beet M14 Line in Prokaryotic Expression System

  • Received:2018-08-29 Revised:2018-12-27 Accepted:2018-10-24 Online:2019-01-28 Published:2019-01-28

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摘要： 为获得纯化的BvM14-STPK蛋白激酶，本研究采用低温16℃，0.5 mmol/L IPTG对转BvM14-STPK的大肠杆菌进行诱导，利用Ni2+亲和层析技术对菌液上清进行纯化，并利用SDS-PAGE对纯化蛋白进行检测。结果表明37℃，0.5 mmol/L IPTG诱导后，BvM14-STPK蛋白以包涵体形式存在，不能对目的蛋白进行纯化分析研究；通过优化诱导条件，确定在低温16℃，0.5 mmol/L IPTG过夜诱导，在上清中获得大量BvM14-STPK可溶性目的蛋白，对菌液上清纯化并经SDS-PAGE检测到BvM14-STPK目的蛋白。在目的蛋白纯化过程中，发现150 mmol/L咪唑洗脱液纯化BvM14-STPK目的蛋白效果最好。本研究成功纯化出目的蛋白BvM14-STPK。

关键词: ‘富士’苹果, ‘富士’苹果, ‘黄金富士’, 采收期, 果实品质, 香气物质

Abstract: To obtain the purified protein kinase BvM14-STPK, E. coli transformed with BvM14-STPK was induced under the condition of low temperature 16℃ and 0.5 mmol/L IPTG in this study. And the supernatant of the bacterium solution was purified by Ni2+ affinity chromatography. Furthermore, SDS-PAGE was applied to detect the purified protein. The result showed that BvM14-STPK protein existed in the form of inclusion bodies after inducing with 0.5 mmol/L IPTG at 37℃ which could not be further purified and analyzed. With better induction conditions, we made sure that a large amount of soluable BvM14-STPK in the supernatant can be obtained after being induced by 0.5 mmol/L IPTG at 16℃ overnight. After the detection with SDS-PAGE to the purification of supernatant, BvM14-STPK protein was detected. During the purification of the target protein, it was found that the imidazole at 150 mmol/L worked the best. The target protein BvM14-STPK was obtained successfully in this study.