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

Liu, Song; Zhang, Dongxu; Wang, Miao; Cui, Wenjing; Chen, Kangkang; Du, Guocheng; Chen, Jian; Zhou, Zhemin

doi:10.1186/1475-2859-10-112

Cited by 37 publications

(34 citation statements)

References 27 publications

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“…Because the pro-peptide is essential for the correct folding of TGase, direct expression of mature TGase yields insoluble inclusion bodies [8] or inactive enzyme [9]. Thus, TGase is usually expressed in a pro-TGase form [10, 11].…”

Section: Introductionmentioning

confidence: 99%

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

Liu

Miao

et al. 2016

BMC Biotechnol

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BackgroundTransglutaminases (TGase), which are synthesized as a zymogen (pro-TGase) in Streptomyces sp., are important enzymes in the food industry. Because this pro-peptide is essential for the correct folding of Streptomyces TGase, TGase is usually expressed in an inactive pro-TGase form, which is then converted to active TGase by the addition of activating proteases in vitro. In this study, Streptomyces hygroscopicus TGase was actively produced by Streptomyces lividans through promoter engineering and codon optimization.ResultsA gene fragment (tg1, 2.6 kb) that encoded the pro-TGase and its endogenous promoter region, signal peptide and terminator was amplified from S. hygroscopicus WSH03-13 and cloned into plasmid pIJ86, which resulted in pIJ86/tg1. After fermentation for 2 days, S. lividans TK24 that harbored pIJ86/tg1 produced 1.8 U/mL of TGase, and a clear TGase band (38 kDa) was detected in the culture supernatant. These results indicated that the pro-TGase was successfully expressed and correctly processed into active TGase in S. lividans TK24 by using the TGase promoter. Based on deletion analysis, the complete sequence of the TGase promoter is restricted to the region from −693 to −48. We also identified a negative element (−198 to −148) in the TGase promoter, and the deletion of this element increased the TGase production by 81.3 %, in contrast to the method by which S. lividans expresses pIJ86/tg1. Combining the deletion of the negative element of the promoter and optimization of the gene codons, the yield and productivity of TGase reached 5.73 U/mL and 0.14 U/mL/h in the recombinant S. lividans, respectively.ConclusionsWe constructed an active TGase-producing strain that had a high yield and productivity, and the optimized TGase promoter could be a good candidate promoter for the expression of other proteins in Streptomyces.

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Section: Introductionmentioning

confidence: 99%

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

Liu

Miao

et al. 2016

BMC Biotechnol

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“…This difference might be caused by the MTG crosslinking ability, which could thicken the cell wall. It has been reported that MTG can thicken the cell wall or outer member to prevent its secretion (29,30). In this work, the MTG activity was detected during the cultivation of the transformants harboring pET-pro-SDB-MTG, pET-pro-SDB-MTG(D1S), and pET-pro-…”

Section: Resultsmentioning

confidence: 81%

“…The MTG from Streptomyces ladakanum could be directly obtained by the coexpression of pro-MTG and a protease in E. coli, and the highest activity was 0.2 U/ml/OD 600 unit (39). In addition, Liu et al revealed that the coexpression of the MTG from S. hygroscopicus with its proregion could secrete mature MTG into the periplasm in E. coli, and the highest activity was 0.13 U/ml/OD 600 unit (30). Meanwhile, they found that it was very difficult to extract active MTG from the periplasm by the osmotic shock method, which might be due to the thickened cell wall caused by MTG's cross-linking ability (30).…”

Section: Sdb-mtg(⌬d1)mentioning

confidence: 99%

“…In addition, Liu et al revealed that the coexpression of the MTG from S. hygroscopicus with its proregion could secrete mature MTG into the periplasm in E. coli, and the highest activity was 0.13 U/ml/OD 600 unit (30). Meanwhile, they found that it was very difficult to extract active MTG from the periplasm by the osmotic shock method, which might be due to the thickened cell wall caused by MTG's cross-linking ability (30). In this study, the MTG activity reached 0.41 U/ml/OD 600 unit.…”

Section: Sdb-mtg(⌬d1)mentioning

confidence: 99%

“…It has been reported that the proregion is a scaffold for packing the mature MTG into a native structure (23,30). The insertion site was speculated to be in a flexible region; thus, each portion (proregion, SDB, and MTG) would have the ability to adjust its relative position to the proper place to produce a functional enzyme.…”

Section: Sdb-mtg(⌬d1)mentioning

confidence: 99%

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pH-Dependent Activation of Streptomyces hygroscopicus Transglutaminase Mediated by Intein

Liu

Cui

et al. 2014

Appl Environ Microbiol

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bMicrobial transglutaminase (MTG) from Streptomyces is naturally secreted as a zymogen (pro-MTG), which is then activated by the removal of its N-terminal proregion by additional proteases. Inteins are protein-intervening sequences that catalyze protein splicing without cofactors. In this study, a pH-dependent Synechocystis sp. strain PCC6803 DnaB mini-intein (SDB) was introduced into pro-MTG to simplify its activation process by controlling pH. The recombinant protein (pro-SDB-MTG) was obtained, and the activation process was determined to take 24 h at pH 7 in vitro. To investigate the effect of the first residue in MTG on the activity and the cleavage time, two variants, pro-SDB-MTG(D1S) and pro-SDB-MTG(⌬D1), were expressed, and the activation time was found to be 6 h and 30 h, respectively. The enzymatic property and secondary structure of the recombinant MTG and two variants were similar to those of the wild type, indicating that the insertion of mini-intein did not affect the function of MTG. This insignificant effect was further illustrated by molecular dynamics simulations. This study revealed a controllable and effective strategy to regulate the activation process of pro-MTG mediated by a mini-intein, and it may have great potential for industrial MTG production.

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

Cited by 37 publications

References 27 publications

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

Improving the active expression of transglutaminase in Streptomyces lividans by promoter engineering and codon optimization

pH-Dependent Activation of Streptomyces hygroscopicus Transglutaminase Mediated by Intein

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

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