The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptideReportar como inadecuado




The order of expression is a key factor in the production of active transglutaminase in Escherichia coli by co-expression with its pro-peptide - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Microbial Cell Factories

, 10:112

First Online: 23 December 2011Received: 26 October 2011Accepted: 23 December 2011

Abstract

BackgroundStreptomyces 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.

ResultsA 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.

ConclusionsOur 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.

KeywordsStreptomyces hygroscopicus transglutaminase pro-peptide co-expression Escherichia coli Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-10-112 contains supplementary material, which is available to authorized users.

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Autor: Song Liu - Dongxu Zhang - Miao Wang - Wenjing Cui - Kangkang Chen - Guocheng Du - Jian Chen - Zhemin Zhou

Fuente: https://link.springer.com/







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