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Molecular modification, expression and purification of new subtype antioxidant peptide from Pinctada fucata by recombinant Escherichia coli to improve antioxidant-activity.

ABSTRACT: The aim of this study was to establish a system for the efficient expression and purification of new subtype of antioxidant peptide from Pinctada fucata meat (NPFMAP), which is designed by molecular modification technology based on the sequence of purified and identified antioxidant peptide from Pinctada fucata meat (PFMAP, Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg), and to better understand the relationship between structure and antioxidant activity. Meanwhile, gene codon usage was optimized and the glutathione S-transferase (GST) tag of pGEX-6P-1 was added to facilitate expression and purification NPFMAP in Escherichia coli. The results of antioxidant activity assay in vitro showed a higher antioxidant activity in NPFMAP than that in enzymatic hydrolysis digested or chemically synthesized PFMAP. In particular, the DPPH scavenging radical activity increased by about 4.7 times after molecular modification. Structural bioanalysis indicated that new subtype antioxidant peptide had spatial conformation and good hydrophilic after modification, which was confirmed by antioxidant activity assays. Thus, the proposed method could be used to obtain NPFMAP with high antioxidant activity.

SUBMITTER: Wu Y

PROVIDER: S-EPMC6133852 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Molecular modification, expression and purification of new subtype antioxidant peptide from Pinctada fucata by recombinant Escherichia coli to improve antioxidant-activity.

Wu Yanyan Y Ma Yongkai Y Li Laihao L Yang Xianqing X

Journal of food science and technology 20180824 10

The aim of this study was to establish a system for the efficient expression and purification of new subtype of antioxidant peptide from Pinctada fucata meat (NPFMAP), which is designed by molecular modification technology based on the sequence of purified and identified antioxidant peptide from Pinctada fucata meat (PFMAP, Gly-Ala-Gly-Leu-Pro-Gly-Lys-Arg-Glu-Arg), and to better understand the relationship betwee ...[more]

PMID: 30228425

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Project description:Pinctada fucata meat is the main by-product of the pearl harvesting industry. It is rich in nutrition, containing a lot of protein and peptides, and holds significant value for both medicine and food. In this study, a new active protein was discovered and expressed heterogeneously through bioinformatics analysis. It was then identified using Western blot, molecular weight, and mass spectrometry. The antibacterial activity, hemolysis activity, antioxidant activity, and Angiotensin-Converting Enzyme II (ACE2) inhibitory activity were investigated. An unknown functional protein was screened through the Uniprot protein database, and its primary structure did not resemble existing proteins. It was an α-helical cationic polypeptide we named PFAP-1. The codon-optimized full-length PFAP-1 gene was synthesized and inserted into the prokaryotic expression vector pET-30a. The induced expression conditions were determined with a final isopropyl-β-d-thiogalactoside (IPTG) concentration of 0.2 mM, an induction temperature of 15 °C, and an induction time of 16 h. The recombinant PFAP-1 protein, with low endotoxin and sterility, was successfully prepared. The recombinant PFAP-1 protein exhibited strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, and the diameter of the inhibition zone was 15.99 ± 0.02 mm. Its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were 37.5 μg/mL and 150 μg/mL, respectively, and its hemolytic activity was low (11.21%) at the bactericidal concentration. The recombinant PFAP-1 protein significantly inhibited the formation of MRSA biofilm and eradicated MRSA biofilm. It also demonstrated potent 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) scavenging activity with a half-maximal inhibitory concentration (IC50) of 40.83 μg/mL. The IC50 of ACE2 inhibition was 5.66 μg/mL. Molecular docking results revealed that the optimal docking fraction of PFAP-1 protein and ACE2 protein was -267.78 kcal/mol, with a confidence level of 0.913. The stable binding complex was primarily formed through nine groups of hydrogen bonds, three groups of salt bridges, and numerous hydrophobic interactions. In conclusion, recombinant PFAP-1 can serve as a promising active protein in food, cosmetics, or medicine.

Dataset Information

Molecular modification, expression and purification of new subtype antioxidant peptide from Pinctada fucata by recombinant Escherichia coli to improve antioxidant-activity.

Publications

Molecular modification, expression and purification of new subtype antioxidant peptide from <i>Pinctada fucata</i> by recombinant <i>Escherichia coli</i> to improve antioxidant-activity.

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