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  

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

作　　者：栗亚美 安展飞 陈阿娜[1,2,3] 杨艳坤[1,2,3] 白仲虎 

机构地区：[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　　处：《生物学杂志》2017年第3期11-17,共7页Journal of Biology

基　　金：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)

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,Ba Pul13A)在大肠杆菌中表达分泌水平的影响,构建了25株Ba Pul13A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/m L,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,48.18U/m L,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Ba Pul13A变体表达分泌水平差异的主要原因。Various tags, especially charged amino acids, were widely applied in soluble expression and secretion of heterogenous pro- teins in Escherichia coil recent years. To know how the charged amino acids types and numbers influent the Pull3A expression and se- cretion, 25 Pull3A 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 Pull3A＇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 secre- tion levels.

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 

分 类 号：Q78[生物学—分子生物学] TQ925[轻工技术与工程—发酵工程]