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

Barbara Salis,Rodolfo Schrepfer,Giancarlo Tonon,Michele Bossi,Silvia Scaramuzza,Gloria Saccani Jotti,Davide Crobu,Gaia Spinetti

doi:10.1186/s12896-015-0202-4

Barbara Salis, Rodolfo Schrepfer + Show 6 more

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https://doi.org/10.1186/s12896-015-0202-4

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Abstract

BackgroundBacterial 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.ResultsThermoinducible 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.ConclusionsCombining 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.

Highlights

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 present a cheaper and more efficient system based on the E.coli peculiarity to highly express recombinant proteins by inclusion body identifying and developing the most performing expression system, the procedure described could potentially be applied for producing pure recombinant microbial transglutaminases (MTGase) on a commercial scale for subsequent use as a convenient biocatalyst for the preparation of new protein conjugates
The long LacZ1–8PNP20MetMTGase fusion protein was expressed in cell strains NP650/1 under constitutive lac promoter and NP656/1 under the thermoinducible phage lambda promoter

Summary

Introduction

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. Transglutaminases (TGases, protein-glutamine γ − glutamyltransferase, E.C.2.3.2.13) are a large family of multifunctional enzymes occurring in several organisms, including mammals, invertebrates, plants and microorganisms. These enzymes catalyze an acyl transfer reaction between the γ-carboxyamide group of a peptidebound glutaminyl residue (acting as acyl donor) and a variety of primary amines (acting as acceptor) including the ε-amino group of a peptide-bound lysine, resulting in the formation of a new γ-glutaminyl covalent link and ammonia [1]. It is widely demonstrated that transglutaminase is involved in several physiological process It serves a key factor in pancreatic fl-cell during glucosestimulated insulin release through Ca2+ dependent enzyme reaction in isolated islets of Langerhans [2]. Transglutaminases can modulate several cardiovascular risk factors, especially hypertension [3]

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