Purification and On-Column Activation of a Recombinant Histidine-Tagged Pro-Transglutaminase after Soluble Expression in<i>Escherichia coli</i>

Yang, Huilin; Pan, Li; Lin, Ying

doi:10.1271/bbb.90422

Cited by 18 publications

(17 citation statements)

References 27 publications

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“…These results suggest that mTGase might be toxic to E. coli , probably due to its protein crosslinking activity. Insoluble mTGase precursor and mTGase can be overexpressed in the cytoplasm of E. coli as inclusion bodies and subsequently solubilized and refolded to yield various amounts of soluble proenzyme or mTGase . Soluble mTGase precursor can be produced in large quantities in the cytoplasm of E. coli by lowering the temperature of protein induction below 37°C .…”

Section: Resultsmentioning

confidence: 99%

“…Soluble mTGase precursor can be produced in large quantities in the cytoplasm of E. coli by lowering the temperature of protein induction below 37°C . Both soluble and inclusion body approaches to produce mTGase precursor, however, necessitate in vitro removal of the pro‐domain to yield active mTGase enzyme by proteases such as TAMEP, Dispase, or Trypsin …”

Section: Resultsmentioning

confidence: 99%

“…Insoluble mTGase precursor and mTGase can be overexpressed in the cytoplasm of E. coli as inclusion bodies and subsequently solubilized and refolded to yield various amounts of soluble proenzyme or mTGase. 21,23 Soluble mTGase precursor can be produced in large quantities in the cytoplasm of E. coli by lowering the temperature of protein induction below 378C. 24,25 Both soluble and inclusion body approaches to produce mTGase precursor, however, necessitate in vitro removal of the pro-domain to yield active mTGase enzyme by proteases such as TAMEP, Dispase, or Trypsin.…”

Section: Design Of Mtgase Precursor-3c Protease Dual Gene Expression mentioning

confidence: 99%

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Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

et al. 2015

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Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/ mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 208C, but results in full activity when shifted to 378C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Design Of Mtgase Precursor-3c Protease Dual Gene Expression mentioning

confidence: 99%

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Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

et al. 2015

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“…The Sn TGase was folded into a "zymogen-like" form, which could subsequently be rendered active by the removal of "proregion-like" RNA binding domain (here, LysRS). We found that LysRS (Choi et al, 2008;Commans et al, 1995;Onesti et al, 2000) was uniquely potent for promoting the solubility and folding of TGase Generally, the pro-region is known to be essential for the soluble expression of TGases of various microbial origin (H. L. Yang et al, 2009;Yu et al, 2008), reflecting its crucial cis-acting role. In addition to TGase, many other proteins are expressed as precursors, and during their folding process, the pro-regions are involved in directly stabilizing the transition state (Baker et al, 1993;Bryan, 2002;Eder & Fersht, 1995).…”

Section: Discussionmentioning

confidence: 96%

“…Typically, the maturation of proTGase into mTGase requires an additional process that cleaves the pro-region by trypsin, dispase, or subtilisin-like protease (Marx, Hertel, & Pietzsch, 2008;Pasternack et al, 1998). However, dispase is too expensive for industrial use (H. L. Yang, Pan, & Lin, 2009) and trypsin is relatively nonspecific and detrimental to TGase (Marx et al, 2008), making it difficult to co-express them in E. coli. Furthermore, co-expressed subtilisin-like protease, SAM-P45, degraded TGase after long-term expression in Corynebacterium glutamicum (Date, Yokoyama, Umezawa, Matsui, & Kikuchi, 2003).…”

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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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Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag

et al. 2011

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In order to produce recombinant microbial transglutaminase (rMTG) which is free of the activating protease, dispase was used to activate the pro-rMTG followed by immobilized metal affinity chromatography (IMAC). As shown by MALDI-MS, the dispase does not only cleave the pro-sequence, but unfortunately also cleaves within the C-terminal histidine-tag. Hence, the active rMTG cannot properly bind to the IMAC material. As an alternative, proteinase K was investigated. This protease was successfully applied for the activation of purified pro-rMTG either as free or immobilized enzyme and the free enzyme was also applicable directly in the crude cell extract of E. coli. Thus, it enables a simple two-step activation/purification procedure resulting in protease-free and almost pure transglutaminase preparations. The protocol has been successfully applied to both, wild-type transglutaminase of Streptomyces mobaraensis as well as to the highly active variant S2P. Proteinase K activates the pro-rMTG without unwanted degradation of the histidine-tag. It turned out to be very important to inhibit proteinase K activity, e.g., by PMSF, prior to protein separation by SDS-PAGE.

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Purification and On-Column Activation of a Recombinant Histidine-Tagged Pro-Transglutaminase after Soluble Expression inEscherichia coli

Cited by 18 publications

References 27 publications

Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

Production of soluble and active microbial transglutaminase inEscherichia colifor site-specific antibody drug conjugation

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

Investigations on the activation of recombinant microbial pro-transglutaminase: in contrast to proteinase K, dispase removes the histidine-tag

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