The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide
<p>Abstract</p> <p>Background</p> <p><it>Streptomyces </it>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 b...
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| Format: | Article |
| Language: | English |
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BMC
2011-12-01
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| Series: | Microbial Cell Factories |
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| Online Access: | http://www.microbialcellfactories.com/content/10/1/112 |
| _version_ | 1818275846128926720 |
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| author | Liu Song Zhang Dongxu Wang Miao Cui Wenjing Chen Kangkang Du Guocheng Chen Jian Zhou Zhemin |
| author_facet | Liu Song Zhang Dongxu Wang Miao Cui Wenjing Chen Kangkang Du Guocheng Chen Jian Zhou Zhemin |
| author_sort | Liu Song |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p><it>Streptomyces </it>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 <it>E. coli</it>. However, expression of pro-TGase by <it>E. coli </it>requires protease-mediated activation <it>in vitro</it>. In this study, we developed a novel co- expression method for the direct production of active TGase in <it>E. coli</it>.</p> <p>Results</p> <p>A TGase from <it>S. hygroscopicus </it>was expressed in <it>E. coli </it>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.</p> <p>Conclusions</p> <p>Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in <it>E. coli</it>, 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.</p> |
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| spelling | doaj.art-2f1bee497769499b805a89328ea588ab2022-12-22T00:09:27ZengBMCMicrobial Cell Factories1475-28592011-12-0110111210.1186/1475-2859-10-112The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptideLiu SongZhang DongxuWang MiaoCui WenjingChen KangkangDu GuochengChen JianZhou Zhemin<p>Abstract</p> <p>Background</p> <p><it>Streptomyces </it>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 <it>E. coli</it>. However, expression of pro-TGase by <it>E. coli </it>requires protease-mediated activation <it>in vitro</it>. In this study, we developed a novel co- expression method for the direct production of active TGase in <it>E. coli</it>.</p> <p>Results</p> <p>A TGase from <it>S. hygroscopicus </it>was expressed in <it>E. coli </it>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.</p> <p>Conclusions</p> <p>Our results indicated that the pro-peptide can assist correct folding of the TGase inter-molecularly in <it>E. coli</it>, 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.</p>http://www.microbialcellfactories.com/content/10/1/112<it>Streptomyces hygroscopicus</it>transglutaminasepro-peptideco-expression<it>Escherichia coli</it> |
| spellingShingle | Liu Song Zhang Dongxu Wang Miao Cui Wenjing Chen Kangkang Du Guocheng Chen Jian Zhou Zhemin The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide Microbial Cell Factories <it>Streptomyces hygroscopicus</it> transglutaminase pro-peptide co-expression <it>Escherichia coli</it> |
| title | The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide |
| title_full | The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide |
| title_fullStr | The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide |
| title_full_unstemmed | The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide |
| title_short | The order of expression is a key factor in the production of active transglutaminase in <it>Escherichia coli </it>by co-expression with its pro-peptide |
| title_sort | order of expression is a key factor in the production of active transglutaminase in it escherichia coli it by co expression with its pro peptide |
| topic | <it>Streptomyces hygroscopicus</it> transglutaminase pro-peptide co-expression <it>Escherichia coli</it> |
| url | http://www.microbialcellfactories.com/content/10/1/112 |
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