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Recombinant production of a bioactive peptide from spotless smooth-hound (Mustelus griseus) muscle and characterization of its antioxidant activity

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Abstract

Bioactive peptides are short amino acid sequences with desirable health effects which are derived from animals, plants, and marine sources. In this study, recombinant production of a bioactive peptide (GIISHR) from spotless smooth-hound (Mustelus griseus) muscle and its antioxidant properties is discussed. A gene composed of 12 tandem copies of the peptide sequence was cloned in pET-28a and expressed as a His-tagged polypeptide in Escherichia coli. The recombinant polypeptide was then purified by Ni–NTA affinity chromatography, cleaved by Trypsin and purified by ultrafiltration. DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS (2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) and hydroxyl radical scavenging activity assays, ferric reducing antioxidant power (FRAP) assay and β-carotene bleaching test were used to characterize the antioxidant activity of the GIISHR. Liquid chromatography–mass spectrometry analysis revealed 60% purity for released bioactive peptide. Production yield was estimated as 60–80 mg GIISHR active peptide per 1 L bacterial culture. Antioxidant activity assays indicated that the antioxidant activity was increased with increase in peptide concentration. Though the DPPH radical scavenging activity, FRAP and β-carotene bleaching power of the peptide were lower than those of the synthetic antioxidant tert-butylhydroquinone (TBHQ), the ABTS and hydroxyl radical scavenging activities of the peptide (at a concentration of 20 mg/mL) were similar to those of TBHQ (at a concentration of 0.1 mg/mL). The findings of the present study may be helpful in development of a process for production of the bioactive antioxidant peptides and its application in food industry.

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Abbreviations

GIISHR:

Gly-Ile-Ile-Ser-His-Arg

DPPH:

1,1-Diphenyl-2-picrylhydrazyl

ABTS:

2,2′-Azinobis-3-ethylbenzotiazoline-6-sulfonic acid

FRAP:

Ferric reducing antioxidant power

TBHQ:

Tert-butylhydroquinone

PCR:

Polymerase chain reaction

BSA:

Bovine serum albumin

TBST:

Tris-buffed saline with Tween

PBS:

Phosphate buffer solution

LC–MS:

Liquid chromatography–mass spectrometry

Prx:

Peroxiredoxin

EM:

Electron migration

HAT:

Hydrogen atom transfer

DNA:

Deoxyribonucleic acid

DAB:

3,3′-Diaminobenzidine

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

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Acknowledgements

This research was funded by of the Biotechnology Development Council of the Islamic Republic of Iran (Grant No: 950901) and conducted at Genetics and Agricultural Biotechnology Institute of Tabarestan.

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Correspondence to Jamshid Farmani.

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Ahmadi-Vavsari, F., Farmani, J. & Dehestani, A. Recombinant production of a bioactive peptide from spotless smooth-hound (Mustelus griseus) muscle and characterization of its antioxidant activity. Mol Biol Rep 46, 2599–2608 (2019). https://doi.org/10.1007/s11033-018-4468-1

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  • DOI: https://doi.org/10.1007/s11033-018-4468-1

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