| Title |
High-level expression of a recombinant active microbial transglutaminase in Escherichia coli
|
|---|---|
| Published in |
BMC Biotechnology, September 2015
|
| DOI | 10.1186/s12896-015-0202-4 |
| Pubmed ID | |
| Authors |
Barbara Salis, Gaia Spinetti, Silvia Scaramuzza, Michele Bossi, Gloria Saccani Jotti, Giancarlo Tonon, Davide Crobu, Rodolfo Schrepfer |
| Abstract |
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. 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. 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. |
X Demographics
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Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United Kingdom | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
The data shown below were compiled from readership statistics for 64 Mendeley readers of this research output. Click here to see the associated Mendeley record.
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| China | 2 | 3% |
| Mexico | 1 | 2% |
| Unknown | 61 | 95% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Master | 14 | 22% |
| Student > Ph. D. Student | 11 | 17% |
| Student > Bachelor | 10 | 16% |
| Researcher | 8 | 13% |
| Student > Postgraduate | 3 | 5% |
| Other | 9 | 14% |
| Unknown | 9 | 14% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 25 | 39% |
| Agricultural and Biological Sciences | 15 | 23% |
| Engineering | 6 | 9% |
| Pharmacology, Toxicology and Pharmaceutical Science | 3 | 5% |
| Arts and Humanities | 2 | 3% |
| Other | 5 | 8% |
| Unknown | 8 | 13% |
Attention Score in Context
This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 16 September 2015.
All research outputs
#20,291,881
of 22,828,180 outputs
Outputs from BMC Biotechnology
#846
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Outputs of similar age
#225,742
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Outputs of similar age from BMC Biotechnology
#29
of 31 outputs
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