Recombinant expression and in vitro activity identification of a bioactive peptide QUB2984 from skin secretion of Agalychnis callidryas

doi:10.12307/2024.445

Abstract

Abstract: BACKGROUND: Frog active peptides have rich activities, such as antibacterial and anti-tumor, and are expected to solve the problem of antibiotic resistance.
OBJECTIVE: The active peptide QUB2984 was discovered in the skin secretions of Agalychnis callidryas. Its structure and properties were simulated by bioinformatics. The peptide was synthesized, purified, and identified and its biological functions were investigated.
METHODS: Agalychnis callidryas skin secretions were collected by electrostimulation. The sequence of QUB2984 was obtained through constructing a cDNA library with isolated mRNA. BLAST was used for peptide sequence alignment. Besides that, Iterative Threading ASSEmbly Refinement (I-TASSER) and HeliQuest tools were used for protein secondary structure simulation. It was synthesized by solid-phase peptide synthesis, purified by reverse-phase high-performance liquid chromatography, and structurally confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The purified peptide was used to evaluate its biological activity. Its antibacterial effect was evaluated by the minimum inhibitory concentration method. Its cytotoxic effect was detected by MTT assay. Its safety was investigated by a hemolysis test.
RESULTS AND CONCLUSION: (1) Peptide QUB2984 had basically α-spiral structure, with a relatively intact hydrophobic surface, and a certain destructive ability to biofilm. The third amino acid position of QUB2984 was composed of W and had a G-X-G structure. (2) The minimum inhibitory concentration of QUB2984 against gram-positive Staphylococcus aureus was 2 μmol/L, the minimum inhibitory concentration against gram-negative Escherichia coli was 2 μmol/L, and the minimum inhibitory concentration against the fungus Candida albicans was 8 μmol/L. (3) The active peptide QUB2984 had obvious inhibitory effect on human non-small cell lung cancer cells NCI-H838 at 10-5 mol/L concentration, and the hemolytic effect on horse red cells at 64 μmol/L concentration was 50%. (4) The results showed that QUB2984 had anti-bacterial and anti-cancer activity, and it had a positive charge of +3, which was conducive to contact with bacteria or cells.

Key words: bioactive peptide, biosynthesis, structural simulation, structure-activity relationship, isolation and purification, antibacterial, anticancer, hemolysis evaluation

CLC Number:

Tang Ziyan, Gu Shunqiang, Chen Xiaoling, Wang Lei, Ma Chengbang, Zhou Mei, Chen Tianbao, Du Lina, Jin Yiguang. Recombinant expression and in vitro activity identification of a bioactive peptide QUB2984 from skin secretion of Agalychnis callidryas[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(17): 2675-2681.

Figures/Tables 5

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