生物学杂志

生物学杂志

• 研究报告 • 上一篇    下一篇

N端9个谷氨酸或7个天冬氨酸显著增强普鲁兰酶在大肠杆菌中表达分泌

  

  1. 1． 江南大学 粮食发酵工艺与技术国家工程实验室， 无锡 214122；
    2． 江南大学 工业生物技术教育部 重点实验室， 无锡 214122； 
    3．江南大学 糖化学与生物技术教育部重点实验室， 无锡 214122
  • 出版日期:2017-06-18 发布日期:2017-06-18
  • 通讯作者: 白仲虎，教授，主要从事发酵工程领域科研工作，E-mail: baizhonghu@jiangnan.edu.cn；杨艳坤，副教授，主要从事发酵工程领域科研工作，E-mail: yangyankun@jiangnan.edu.cn
  • 作者简介:栗亚美，硕士研究生，主要从事发酵工程研究，E-mail: 1198066405@qq.com
  • 基金资助:
    中央高校基本科研业务费专项资金资助(JUSRP51401A)；863项目（2015AA020802）；973项目（2013CB733602）； 国家自然科学基金项目（31570034）

N-terminal 9-glutamine and 7-aspartate tags significantly increase the expression and secretion of pullulanase from Bacillus acidopullulyticus in Escherichia coli

  1. 1. National Engineering Laboratory for Cereal Fermentation Technology; 2. The Key Laboratory of Industrial Biotechnology, Ministry of Education; 3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
  • Online:2017-06-18 Published:2017-06-18

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摘要： 各种标签被广泛应用于大肠杆菌分泌表达异源蛋白，其中最简单有效地便是氨基酸标签，尤其是带电荷氨基酸标签。为了研究N端添加不同种类和数目的带电荷氨基酸标签对普鲁兰酶(pullulanase, BaPul13A)在大肠杆菌中表达分泌水平的影响，构建了25株BaPul13A变体。实验结果表明：N端添加9个连续的谷氨酸标签，降低了mRNA的转录水平，增加了N端mRNA的稳定性，使得普鲁兰酶总酶活达到179.60 U/mL，比不添加氨基酸标签的对照菌高278.28%。N端添加7个连续的天冬氨酸标签，增强了大肠杆菌细胞内膜通透性，大量的普鲁兰酶，48.18 U/mL，分泌到培养基中。通过蛋白质结构预测软件I-TASSER分析得知，N端添加不同带电荷氨基酸标签引起CBM41结构域发生变化，猜测该变化是导致BaPul13A变体表达分泌水平差异的主要原因。

关键词: 带电荷氨基酸标签, 表达分泌, mRNA, 膜通透性, 蛋白结构预测

Abstract: Various tags, especially charged amino acids, were widely applied in soluble expression and secretion of heterogenous proteins in Escherichia coli recent years. To know how the charged amino acids types and numbers influent the Pul13A expression and secretion, 25 Pul13A mutants were constructed, which had different N-terminal amino acid tags. Among them, the nine-glutamine tag could decrease the transcription level and increase the N-terminal mRNA stability, and Pul13A′s activity changed to 179.60 U/mL that was 278.28% higher than the control without any amino acid tag. Meanwhile, the N-terminal seven-aspartate tag enhanced the inner membrane permeability, and secreted massive pullulanse into the culture medium ( 48.18 U/mL ). Furthermore, it was found that the CBM41 structural domain was different in different mutants, which maybe mainly result in the differences of the expression and secretion levels.

Key words: charged-amino-acid tags, expression and secretory, mRNA, membrane permeability, protein structure prediction