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

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dc.contributor.author Salis, Barbara
dc.contributor.author Spinetti, Gaia
dc.contributor.author Scaramuzza, Silvia
dc.contributor.author Bossi, Michele
dc.contributor.author Saccani Jotti, Gloria
dc.contributor.author Tonon, Giancarlo
dc.contributor.author Crobu, Davide
dc.contributor.author Schrepfer, Rodolfo
dc.date.accessioned 2015-09-17T11:28:24Z
dc.date.available 2015-09-17T11:28:24Z
dc.date.issued 2015-09-15
dc.identifier.issn 1472-6750
dc.identifier.uri http://hdl.handle.net/11050/1149
dc.description.abstract Background: 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.language.iso en IT
dc.publisher BioMed Central IT
dc.relation.ispartof BMC Biotechnology IT
dc.relation.ispartofseries 15;84
dc.rights Attribuzione - Non commerciale - Condividi allo stesso modo 3.0 Italia *
dc.rights.uri http://creativecommons.org/licenses/by-nc-sa/3.0/it/ *
dc.subject MTGase IT
dc.subject Escherichia coli IT
dc.subject proteins IT
dc.subject microbial transglutaminase IT
dc.subject Streptomyces mobaraensis IT
dc.title High-level expression of a recombinant active microbial transglutaminase in Escherichia coli IT
dc.type Articolo IT
dc.description.status Pubblicato IT
dc.identifier.doi 10.1186/s12896-015-0202-4 IT
dc.subject.een-cordis EEN CORDIS::SCIENZE BIOLOGICHE ::Biologia / biotecnologia ::Biologia cellulare e molecolare IT

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Attribuzione - Non commerciale - Condividi allo stesso modo 3.0 Italia Attribuzione - Non commerciale - Condividi allo stesso modo 3.0 Italia