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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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