High-level expression of a recombinant active microbial transglutaminase in Escherichia coli

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dc.contributor.authorSalis, Barbara
dc.contributor.authorSpinetti, Gaia
dc.contributor.authorScaramuzza, Silvia
dc.contributor.authorBossi, Michele
dc.contributor.authorSaccani Jotti, Gloria
dc.contributor.authorTonon, Giancarlo
dc.contributor.authorCrobu, Davide
dc.contributor.authorSchrepfer, Rodolfo
dc.date.accessioned2015-09-17T11:28:24Z
dc.date.available2015-09-17T11:28:24Z
dc.date.issued2015-09-15
dc.description.abstractBackground: Bacterial transglutaminases are increasingly required as industrial reagents for in vitro modification of proteins in different fields such as in food processing as well as for enzymatic site-specific covalent conjugation of therapeutic proteins to polyethylene glycol to get derivatives with improved clinical performances. In this work we studied the production in Escherichia coli of a recombinant transglutaminase from Streptomyces mobaraensis (microbial transglutaminase or MTGase) as enzymatically active chimeric forms using different expression systems under the control of both lac promoter or thermoinducible phage lambda promoter. Results: Thermoinducible and constitutive expression vectors were constructed expressing Met-MTGase with chimeric LacZ 1-8 PNP 1–20 or LacZ 1–8 fusion protein under different promoters. After transformed in competent Escherichia coli K12 strains were fermented in batch and fed-bach mode in different mediums in order to select the best conditions of expression. The two most performing fusion protein systems namely short thermoinducible LacZ 1–8 Met-MTGase from NP668/1 and long constitutive LacZ 1-8 PNP 1–20 Met-MTGase from NP650/1 has been chosen to compare both efficiency of expression and biochemical qualities of the product. Proteins were extracted, purified to homogeneity and verified as a single peak obtained in RP-HPLC. The LacZ 1-8 PNP 1–20 Met-MTGase fusion protein purified from NP650/1 exhibited an activity of 15 U/mg compared to 24 U/mg for the shorter fusion protein purified from NP668/1 cell strain. Conclusions: Combining the experimental data on expression levels and specific activities of purified MTGase fusion proteins, the chimeric LacZ 1-8 Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.IT
dc.description.statusPubblicatoIT
dc.identifier.doi10.1186/s12896-015-0202-4IT
dc.identifier.issn1472-6750
dc.identifier.urihttp://hdl.handle.net/11050/1149
dc.language.isoenIT
dc.publisherBioMed CentralIT
dc.relation.ispartofBMC BiotechnologyIT
dc.relation.ispartofseries15;84
dc.rightsAttribuzione - Non commerciale - Condividi allo stesso modo 3.0 Italia*
dc.rights.urihttp://creativecommons.org/licenses/by-nc-sa/3.0/it/*
dc.subjectMTGaseIT
dc.subjectEscherichia coliIT
dc.subjectproteinsIT
dc.subjectmicrobial transglutaminaseIT
dc.subjectStreptomyces mobaraensisIT
dc.subject.een-cordisEEN CORDIS::SCIENZE BIOLOGICHE ::Biologia / biotecnologia ::Biologia cellulare e molecolareIT
dc.titleHigh-level expression of a recombinant active microbial transglutaminase in Escherichia coliIT
dc.typeArticoloIT
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