High-level Expression of the Chemically Synthesized Gene for Microbial Transglutaminase from <i>Streptoverticillium</i> in <i>Escherichia coli</i>

Kawai, Misako; Takehana, Shino; Takagi, Hiroshi

doi:10.1271/bbb.61.830

Cited by 26 publications

(21 citation statements)

References 5 publications

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“…Many studies have shown that the pro-peptide is essential for overexpression of soluble TGase in E. coli [9,11,17,18]. To investigate the effects of the secretory signal peptide and the covalently linked pro-peptide on expression of S. hygroscopicus TGase in E. coli , the pTG encoding TGase alone, the pSTG encoding TGase fused with the pelB signal peptide, and the pSPTG encoding pro-TGase fused with the pelB signal peptide were constructed and used for TGase expression (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, three strategies have been used for the expression of the microbial TGase in E. coli : (i) the direct expression of TGase fused or not fused to an additional peptide; (ii) the expression of pro-TGase followed by processing to TGase in vitro ; and (iii) the co-expression of pro-TGase with the activating protease. The first strategy often leads to a low-level of the protein expression or the inclusion body formation [9,10]. The second strategy produces a large amount of soluble pro-TGase [11,12] that can be converted into an active TGase in vitro by adding exogenous proteases [13].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2011

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“…Moreover, TGase has been utilized in nonfood industries, such as protein engineering, textile and leather processing, pharmaceutical industries, and development of surgical materials (Fontana, Spolaore, Mero, & Veronese, ; Y. Liu et al, ; Rachel, Quaglia, Levesque, Charette, & Pelletier, ; Zhu & Tramper, ). Among the many microbial TGases that have been studied for their expression and activity in Escherichia coli , Streptomyces mobaraensis TGase (Sm TGase) is the first industrial enzyme (Kawai, Takehana, & Takagi, ; Pasternack et al, ; Yokoyama, Nakamura, Seguro, & Kubota, ).…”

Section: Introductionmentioning

confidence: 99%

RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

Lee

Son

Kim

et al. 2019

Biotech & Bioengineering

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Transglutaminase (TGase) induces the cross‐linking of proteins by catalyzing an acyl transfer reaction. TGase is a zymogen, activated by the removal of its pro‐region. Because the pro‐region is crucial for folding and inhibition of the TGase activity, the recombinant expression of the mature TGase (mTGase) without the pro‐region, usually results in inactive inclusion bodies or low protein yield. Here, Streptomyces netropsis TGase was fused with Escherichia coli lysyl‐tRNA synthetase (LysRS), as a module with chaperoning activity in an RNA dependent manner (chaperna). The TGase activity from purified fusion protein induced via the removal of LysRS by tev protease in vitro. Moreover, active mTGase was produced in E. coli via an intracellular cleavage system, wherein LysRS‐mTGase was cleaved by the coexpressed tev protease in vivo. The results suggest that LysRS essentially mimics pro‐region, which exerts a dual function—folding of TGase into active conformation and keeping it as dormant state—in an RNA‐dependent manner. Thus, trans‐acting RNAs, prompt the cis‐acting chaperone function of LysRS, while being mechanistically similar to the intramolecular chaperone function of the pro‐region. These results could be implemented and extended for the folding of “difficult‐to‐express” recombinant proteins, by harnessing the chaperna function.

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“…S-8112 TGase (microbial transglutaminase, MTG) was obtained from the E. coli expression system described previously and was purified by ion-exchange FPLC for preparation (21,22). Bovine ␣-lactalbumin, cysteine-sulfated insulin A-chain, cysteine-sulfated insulin B-chain, trypsinogen, carbonic anhydrase, ovalbumin, bovine serum albumin (BSA), and lysozyme were purchased from Sigma Chemical Co.…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Labeling of Arbitrary Proteins

Shimba

Yamada

Yokoyama

et al. 2002

Analytical Biochemistry

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High-level Expression of the Chemically Synthesized Gene for Microbial Transglutaminase from Streptoverticillium in Escherichia coli

Cited by 26 publications

References 5 publications

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

RNA‐dependent chaperone (chaperna) as an engineered pro‐region for the folding of recombinant microbial transglutaminase

Enzymatic Labeling of Arbitrary Proteins

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