1. Molecular cloning, expression, purification, and functional characterization of palustrin-2CE, an antimicrobial peptide of Rana chensinensis
Yan Sun, Qian Li, Zhi Li, Yuan Zhang, Jie Zhao, Lin Wang Biosci Biotechnol Biochem. 2012;76(1):157-62. doi: 10.1271/bbb.110672. Epub 2012 Jan 7.
Antimicrobial peptides are effector molecules of the innate immunity of amphibians. Here, one antimicrobial peptide cDNA precursor, prepropalustrin-2CE3, from the tadpole of the Chinese brown frog Rana chensinensis was cloned. The coding sequence corresponding to the mature palustrin-2CE peptide was subcloned into pGEX-6p-1. The soluble GST-palustrin-2CE fusion protein was successfully expressed in the BL21(DE3)pLysS strain at 16 °C, and the proportion of the fusion protein reached 35%-39% of the total cellular protein. After removal of the GST-fusion tag, the purity of the palustrin-2CE obtained by Sephadex G50 chromatography was about 97%. Moreover, the purified palustrin-2CE displayed obviously inhibitory activities against the sensitive bacteria Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and multi-drug resistant S. aureus and E. coli. These findings suggest that the tadpole of the Chinese brown frog is a unique source of antimicrobial peptides and indicates the therapeutic potential of the palustrin-2CE peptide.
2. Molecular cloning of novel antimicrobial peptide genes from the skin of the Chinese brown frog, Rana chensinensis
Jie Zhao, Yan Sun, Zhi Li, Qi Su Zoolog Sci. 2011 Feb;28(2):112-7. doi: 10.2108/zsj.28.112.
One species of the Chinese brown frog, Rana chensinensis, is widely distributed in north-central China. In this study, a cDNA library was constructed to clone the antimicrobial peptides' genes from the skin of R. chensinensis. Twenty-three prepropeptide cDNA sequences encoding twelve novel mature antimicrobial peptides were isolated and characterized. Six peptides belonged to three known families previously identified from other Ranid frogs: temporin (4 peptides), brevinin-2 (1 peptide), and palustrin-2 (1 peptide). The other six peptides showed little similarity to known antimicrobial peptides. According to the amino acid sequences, with or without α-helix structure, and either hydrophilic or hydrophobic, these were organized into four new families: chensinin-1 (3 peptides), chensinin-2 (1 peptide), chensinin-3 (1 peptide), and chensinin-4 (1 peptide). Five peptides from different families were chemically synthesized, and their antimicrobial, cytolytic, and hemolytic activities were evaluated. Of these, brevinin-2CE showed strongest antimicrobial activities against both the Gram-positive and Gram-negative bacteria with a slight hemolysis. Temporin-1CEe and palustrin-2CE also displayed a slight hemolysis, but they had different activities to prokaryotic cells. Temporin-1CEe showed higher antimicrobial activity against Gram-positive bacteria than Gram-negative bacteria, whereas it was contrary to palustrin-2CE. Chensinin-1 CEb and chensinin-3CE only had moderate antimicrobial activity against microorganisms. In addition, the brevinin-2 peptides from different brown frogs were analyzed to reveal the taxonomy and phylogenetic relationships of R. chensinensis.
