The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide.
The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide.
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表达顺序是大肠杆菌通过与其前肽共表达产生活性转谷氨酰胺酶的关键因素
DOI：
10.1186/1475-2859-10-112
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发表时间：
2011-12-23
影响因子：
6.4
通讯作者：
Zhou Z
中科院分区：
文献类型：
--
作者：
Liu S;Zhang D;Wang M;Cui W;Chen K;Du G;Chen J;Zhou Z
背景
链霉菌转氨酶（TGase）是天然合成的酶原（pro-TGase），然后通过去除其N-末端前肽来加工以产生活性酶。发现该前肽对于可溶性TGase在E.杆菌而原TGase在E.大肠杆菌需要蛋白酶介导的体外活化。在本研究中，我们发展了一种新的共表达方法，在大肠杆菌中直接生产活性TGase。杆菌
结果
从S. hygroscopicus在E. pelB信号肽与pelB融合后，pelB蛋白酶活性下降，但pelB信号肽与pelB融合后，pelB蛋白酶活性下降，pelB信号肽与pelB融合后，pelB蛋白酶活性下降，但pelB信号肽与pelB信号肽融合后，pelB蛋白酶活性下降。因此，通过使用载体pET-22 b（+）在单个T7启动子下共表达TGase及其前肽作为独立多肽来设计替代方案。虽然前肽共表达，但在重组细胞的可溶性和不溶性级分中均检测不到与信号肽融合的TGase。类似地，当两种基因以TGase和前肽的顺序表达时，与信号肽融合的TGase的溶解性没有通过与其前肽的共表达而改善。有趣的是，活性TGase仅由其中前肽和TGase与信号肽融合并顺序表达的细胞产生。纯化的重组和天然TGase具有相似的催化性质。
结论
我们的结果表明，前肽可以帮助正确折叠的TGase分子间在E。在大肠杆菌中，前肽的表达先于TGase的表达是产生活性TGase的必要条件。基于优化基因表达顺序的共表达策略可用于作为前体合成的其他功能蛋白的表达。
Background
Streptomyces transglutaminase (TGase) is naturally synthesized as zymogen (pro-TGase), which is then processed to produce active enzyme by the removal of its N-terminal pro-peptide. This pro-peptide is found to be essential for overexpression of soluble TGase in E. coli. However, expression of pro-TGase by E. coli requires protease-mediated activation in vitro. In this study, we developed a novel co- expression method for the direct production of active TGase in E. coli.
Results
A TGase from S. hygroscopicus was expressed in E. coli only after fusing with the pelB signal peptide, but fusion with the signal peptide induced insoluble enzyme. Therefore, alternative protocol was designed by co-expressing the TGase and its pro-peptide as independent polypeptides under a single T7 promoter using vector pET-22b(+). Although the pro-peptide was co-expressed, the TGase fused without the signal peptide was undetectable in both soluble and insoluble fractions of the recombinant cells. Similarly, when both genes were expressed in the order of the TGase and the pro-peptide, the solubility of TGase fused with the signal peptide was not improved by the co-expression with its pro-peptide. Interestingly, active TGase was only produced by the cells in which the pro-peptide and the TGase were fused with the signal peptide and sequentially expressed. The purified recombinant and native TGase shared the similar catalytic properties.
Conclusions
Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in E. coli, and expression of pro-peptide prior to that of TGase was essential for the production of active TGase. The co-expression strategy based on optimizing the order of gene expression could be useful for the expression of other functional proteins that are synthesized as a precursor.
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影响因子：
4.4
作者：
Fu, RY;Chen, J;Li, Y
通讯作者：
Li, Y
吸水链霉菌的两种不同蛋白酶参与转谷氨酰胺酶激活
影响因子：
6.1
作者：
Zhang, Dongxu;Wang, Miao;Chen, Jian
通讯作者：
Chen, Jian
影响因子：
4.4
作者：
Kikuchi, Y;Date, M;Matsui, H
通讯作者：
Matsui, H
影响因子：
1.6
作者：
Tan, S
通讯作者：
Tan, S
影响因子：
8.8
作者：
Cui, Li;Du, Guocheng;Chen, Jian
通讯作者：
Chen, Jian