title: High-level expression of a recombinant active microbial transglutaminase in Escherichia coli.
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 LacZ1-8PNP1-20 or LacZ1-8 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 LacZ1-8Met-MTGase from NP668/1 and long constitutive LacZ1-8PNP1-20Met-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 LacZ1-8PNP1-20Met-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 LacZ1-8Met-MTGase, which displays an enzymatic activity comparable to the wild-type enzyme, was selected as a candidate for producing microbial transglutaminase for industrial applications.
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy. barbara.salis@multimedica.it.
authors.forename: Barbara
authors.initials: B
authors.lastname: Salis
authors.affiliation: IRCCS MultiMedica, Milan, Italy.
authors.forename: Gaia
authors.initials: G
authors.lastname: Spinetti
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Scaramuzza
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Bossi
authors.affiliation: Department of Biomedical, Biotechnological and Traslational Science (S.Bi.Bi.T.), University of Parma, Via Volturno 39, 43121, Parma, Italy.
authors.forename: Gloria
authors.initials: G
authors.lastname: Saccani Jotti
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Tonon
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Crobu
authors.affiliation: Bio-Ker S.r.l., Sardinia Scientific and Technological Park, Building 3, 09010, Pula, Cagliari, Italy.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Schrepfer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005285:Fermentation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D011503:Transglutaminases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D011503:Transglutaminases