별불가사리(Asterina pectinifera) 유래의 cysteine rich 항균활성펩타이드의 분자적 특성과 재조합 펩타이드 생산에 관한연구

Abstract

Antimicrobial peptides (AMPs) are innate immune factors distributed among most life forms including animals, plants, and bacteria. Marine invertebrates like starfish, Asterina pectinifera, depend on these factors for their survival in the microbe-rich seawater and marine sediments due to lack of adaptive immune system. To this day, there has been no reports of AMPs in this starfish species. In this study, the cDNA cloning, genomic structure analysis, recombinant production, antibacterial activity assay, and transcriptional expression of previously isolated starfish AMP, Asterocin from the tube feet of P. pectinifera are reported. cDNA cloning revealed that Asterocin comprises 1078 nucleotides including a 3’ untranslated region (UTR) of 78 bp, an open reading frame (ORF) of 234 bp, and a 5’ UTR of 766 bp. The ORF encoded a preproprotein of 77 amino acids (AAs), which was composed of a signal peptide of 19 AAs, prosequence of 24 AAs, a mature peptide of 33 AAs, and one Leu residue at the C-terminal end that is removed in the post-translational modification process. The mature peptide contained 4 cysteine residues that form 2 disulfide bonds. The homology search using the mature amino acid sequence of Asterocin revealed that Asterocin had no significant homology to AMPs from other species. However, the pattern of Asterocin gene structure, which was two exons separated by one intron, was similar to the gene structure of other echinoderm AMPs. Antimicrobial activity assay demonstrated Asterocin was active regardless of its disulfide bond formation against both Gram-positive and Gram-negative bacteria. However, the most potent antimicrobial activity of Asterocin was observed when its disulfide bonds were formed. The transcriptional expression level of Asterocin was high in tube feet and coelomic epithelium. However, the expression level did not show statistically significant change when these tissues were challenged with Bacillus subtilis. Collectively, Asterocin is a novel cysteine-rich AMP from echinoderm that needs disulfide bond formation in the post-translational process for potent antimicrobial activity.

Peptides are small polymers typically composed of 50 or less amino acids. Although numerous bioactive peptides have been reported up to date, mass production of peptides are often difficult limiting their utilization in pharmaceutical or cosmetic industries. This study aims to propose an effective and economic mass production method for these bioactive peptides. The expression plasmid is constructed firstly by fusing Thioredoxin A sequence into pET28a(+) vector, then the nucleotide sequences of desired peptides with Met at the beginning were inserted using restriction enzyme sites in the multiple cloning site (pET28a(+)-TrxA-peptides). The plasmid was transformed into Escherichia coli BL21 (DE3) and overexpression of the recombinant fusion protein was induced. Then, each peptide is separated by cyanogen bromide (CNBr) digestion, which cleaves C-terminus of Met residues. This method allows the simultaneous production of different peptides. Moreover, Met changes in the CNBr digestion leaving a homoserine lactone at the C-terminus contributing to structural stability of recombinant peptide.