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High-level expression of a recombinant active microbial transglutaminase in Escherichia coli
Saturday, 2015/09/26 | 05:50:18

Barbara Salis1*, Gaia Spinetti2, Silvia Scaramuzza1, Michele Bossi1, Gloria Saccani Jotti3, Giancarlo Tonon1, Davide Crobu1 and Rodolfo Schrepfer1

 

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.

 

Fig. 4. Reverse-phase (a) and size-exclusion HPLC (b) analysis of purified MTGase from NP668

Salis et al. BMC Biotechnology 2015 15:84   doi:10.1186/s12896-015-0202-4

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