**6. Isolation and discovery of cationic antimicrobial peptides**

#### **6.1. Traditional biochemical purification**

Standard biochemical techniques for isolation of small peptides have been used to isolate CAPs from tissues, skin secretions and other biological fluids. Briefly, tissue lysates or fluids are diluted in 0.1% trifluoroacetic acid (TFA) and extracted under ice-cold acid conditions. After centrifugation, the acidic supernatant is loaded on a C18 column and the peptide elut‐ ed with increasing acetonitrile (5-80%) in 0.05% TFA. Those fractions with antimicrobial ac‐ tivity are pooled and further purified by reverse-phase HPLC with increasing acetonitrile (5-55%) in 0.05% TFA. The material in absorbance peaks is then pooled, dried under vacuum and reconstituted in MilliQ water for mass and sequence analysis using mass spectrometry and automated Edman degradation.

Pleurocidin was originally isolated from homogenates of the skin and mucus secretions of winter flounder using C18 chromatography followed by size fractionation on Sephadex G-50 (Cole et al., 1997). Biologically active fractions were pooled and subjected to strong cat‐ ion exchange HPLC and then reversed phase HPLC.

#### **6.2.Genomics approaches to antimicrobial peptide discovery**

Once even a partial amino acid sequence of a CAP is known, genomic strategies can be em‐ ployed to identify and isolate DNA sequences encoding CAPs. This can involve screening of cDNA and genomic libraries using oligonucleotide probes or amplification of CAP-encod‐ ing sequences from genomic or cDNA by PCR (Iwamuro and Kobayashi, 2010). CAP-encod‐ ing sequences can also be identified in genomic and EST sequencing databases by similarity searching using bioinformatics search engines such as BLAST (Browne et al., 2011; Fer‐ nandes et al., 2010). Full-length cDNA sequences can then be obtained by 5'- and 3'-RACE. The identification of 28 new human and 43 new mouse β-defensin genes was achieved using a computational search tool to find conserved motifs in draft genome sequences (Schutte et al., 2002).

Pleurocidin was originally cloned from cDNA and genomic libraries using degenerate oligo‐ nucleotide probes (Cole et al., 2000; Douglas et al., 2001) or PCR amplicons encoding indi‐ vidual pleurocidin variants (Douglas et al., 2003). Additional pleurocidin variants were amplified by PCR from cDNA of different flatfish species using primers corresponding to the conserved amino-terminal signal peptide and carboxy-terminal anionic propiece (Patr‐ zykat et al., 2003). At least 30 variants have now been cloned (Figure 3).

#### **6.3.** *In silico* **discovery and rational peptide design**

Various *in silico* methods have been used to rationally design CAPs and predict their biolog‐ ical activity (Hadley and Hancock, 2010). These include the use of artificial neural networks, support vector machines and quantitative structure activity relationship (QSAR) modeling (Fjell et al., 2011; Torrent et al., 2011). Combinatorial synthesis and high throughput peptide synthesis using SPOT technology (Hilpert et al., 2007) together with a QSAR-based artificial neural network system (Fjell et al., 2009) have also been used to design active synthetic CAPs. This approach, coupled with high-throughput screening assays using peptide arrays, is invaluable for inexpensively generating large numbers of potential candidates for clinical assessment (Cherkasov et al., 2009).
