## 4 Discussion  
By means of genetic engineering, desired antimicrobial peptide proteins are generated by several systems including eukaryotic systems such as the _E. coli_ expression system [43] and the baculovirus expression vector system [39], and prokaryotic systems such as the yeast expression system [44; 45]. _E. coli_ is the least expensive and most efficient expression system for small proteins [40]. Advantages include rapid bacterial growth, quick expression and high product yields [46]. Several antibacterial peptides with biological activities have been expressed successfully by the _E. coli_ expression system. According to the reports, Beta-defensin-4 from human [47; 48], bovine LfcinB [36], porcine ecropin P1 [49], sarcotoxin IA from _Sarcophaga peregrina_ larva [50] and adenoregulin from arboreal frog (_Phylomedusa bicolor_) [51] were expressed with an _E. coli_ expression system. Among aquatic species, previous reports have revealed that hepcidin from Tilapia (_Oreochromis mosamalicus_) [52] and channel catfish [42], Piscidin-1 from Striped bass (_Morone saxatilis_) [53], LEAP-2 from Grass carp (_Ctenopharyngodin idella_) [54] have been successfully expressed via an _E. coli_ expression system. In our study, three mNKLs were expressed via an _E. coli_ expression system and produced the approximately 24.6 kDa Trx-His-mNKLs. After purification, we finally received the concentration of mNKL 1 at 1.1 mg/mL, mNKL 2 at 1.4 mg/mL and mNKL 3 at 1.7 mg/mL. Previous studies have shown that AMPs have high toxicity towards host strains. Therefore, different molecular chaperones are used in order to reduce toxicity and inclusion bodies to improve fusion protein expression [27]. The pET-32a(+) plasmid vector includes the Trx.Tag(tm) thioredoxin protein which is widely used as a fusion molecular chaperone for protein expression in _E. coli_[42]. The thioredoxin can dramatically increase the solubility of heterologous proteins synthesized in the _E. coli_ cytoplasm and reduce the toxicity towards host strains, and the thioredoxin fusion proteins usually accumulate to a high level [50; 55]. However, the thioredoxin fusion proteins will impress the activity of recombined AMPs. Enterokinase can specially recognize the amino acid sequence DDDDK and digest the peptide bond after the lysine residue. This is particularly important to get mature short peptides in the _E. coli_ expression system such as mNKLs that are only 76 residues and account for only 34.9% of the fusion protein. In order to get a higher level of expression and soluble protein, different induction temperatures (25 \({}^{\circ}\)C, 30 \({}^{\circ}\)C and 37 \({}^{\circ}\)C), IPTG concentrations (0.1 mM to 1.0 mM) and induction times (1 h to 24 h) were optimized for recombinant mNKLs expression. In addition, a higher level of expression of fusion soluble Trx-His-mNKLs (approximately 24.6 kDa) was induced by 0.1 mM IPTG for 4 h at 37 \({}^{\circ}\)C. Subsequently, the mNKLs (approximately 8.6 kDa) were cleaved with Recombinant Bovine Enterokinase Light Chain-His and the enterokinase, Trx-His tag and unseparated Trx-His-mNKLs proteins were removed by the Ni-NTA-Sefinose column. However, the production of cleaved mNKLs was very low (approximately 0.1 mg/mL). In recent years, an increasing number of antimicrobial peptides have been identified and extensive research has been conducted for the analysis of their structures and antimicrobial activity. Although some have also been reported to have potent activities against other microorganisms such as fungi, protozoa, and parasites [1; 2; 4; 56], antibacterial activity is still the primary function of antimicrobial peptides. Although studies on the antibacterial mechanism of AMPs have been conducted widely, the precise mechanism of action is not fully understood. Regardless of their precise mode of action, AMPs consistently demonstrate antibacterial activity by interacting with bacterial cell membranes [57]. Similar to other members of the SAPLIP family, NK-lysins posses the general ability to interact with lipids [21].