LAB of Genome Biology > RESEARCH > Antimicrobial Peptides
We are identifying and analyzing antimicrobial peptides(AMP) as antibiotics alteratives and developing a method for a large scale production of AMPs.
1) Identification and analysis of animicrobial peptides(AMP) as antibiotics alternatives
In this study, we characterized 29 functional β-defensin genes in the pig genome on the basis of sequence homology to previously reported β-defensin genes and the conserved 6-cysteine motif. We compared the evolutionary conservation of β-defensin genes among humans, cattle, and pigs, and analyzed their expression patterns. We also report SNPs in the mature peptide region of porcine β-defensin genes. [PMID: 23150902]
2) Development of a method for a large scale production of antimicrobial peptides
Growing number of multidrug antibiotic resistant pathogenic microorganism is hugely considered as a serious issue in the health industry. Recently, antimicrobial peptides (AMPs) received substantial interest due to their high activity against the board range of pathogenic microorganism including multidrug resistant microorganisms. However, due to their extreme toxicity and proteolytic degradation, the mass production of AMPs by conventional heterologous expression system is still facing challenges to be addressed. Productions of these highly toxic proteins such as short AMPs in Escherichia coli, several methods such as insoluble and soluble fusion partner systems were available. However, these strategies have serious limitation in terms of high yield.
Recently, we have developed a highly simple, robust, and cost-effective method for the large-scale production of these bacteriotoxic peptides such as AMPs in E. coli. We simply inserted the AMPs into the scaffold of the engineered Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield (Figure.), circumventing the inherent toxicity and proteolytic degradation of AMP production in E. coli. Indeed, we were able to successfully demonstrated that this method which can achieve high efficiency production at large-scale for biologically active antimicrobial peptides and other bacteriotoxic proteins for variety of application.
Figure. AMPs are cloned into a loop region of methionine-free GFP (r5M-172-AMP-173) and expressed as insoluble proteins in E. coli. The insoluble proteins are extracted and purified by Ni-NTA and cleaved at methionine sites using cyanogen bromide. Finally, AMPs are purified by high performance liquid chromatography and refolded to their biologically native structures. [PMID: 26864123]
3) AMP expression patterns in Pig
Figure. Expression patterns of novel protegrins in multiple pig tissues
Protegrins (PGs) are potent antimicrobial peptides that act on a broad spectrum of microorganisms, including bacteria, fungi and some enveloped viruses. We analyzed the expression pattern of protegrins in 17 different pig tissues using RT–PCR, and developed an anti-(PG–1) polyclonal IgG. Western blot analysis using the antibody showed that protegrins are mainly present as prepropeptide forms in normal tissues, rather than as mature peptides. Immunohistochemical analysis showed that protegrin expression was specific to a few cell types, including neutrophils, pulmonary club, epithelial and Leydig cells. Genetic analyses of the five previously reported protegrin sequences showed that they are encoded at a single locus, rather than from multiple paralogous genes. By genotyping 28 animals across five breeds, we identified eight different alleles of the PGs. [PMID: 25264901]