Antimicrobial peptides (AMPs) are part of the innate immune system and are generally defined as cationic, amphipathic peptides, with less than 50 amino acids, including multiple arginine and lysine residues. The human cathelicidin antimicrobial peptide LL37 can be found at different concentrations in many different cells, tissues and body fluids and has a broad spectrum of antimicrobial and immunomodulatory activities. The healing of wound is a complex process that involves different steps: hemostasis, inflammation, remodeling/granulation tissue formation and re-epithelialization. Inflammation and angiogenesis are two fundamental physiological conditions implicated in this process. We have recently developed a new method for the expression and purification of recombinant LL37. In this work, we show that the recombinant peptide P-LL37 with a N-terminus proline preserves its immunophysiological properties in vitro and in vivo. P-LL37 neutralized the activation of macrophages by lipopolysaccharide (LPS). Besides, the peptide induced proliferation, migration and tubule-like structures formation by endothelial cells. Wound healing experiments were performed in dexamethasone-treated mice to study the effect of LL37 on angiogenesis and wound regeneration. The topical application of synthetic and recombinant LL37 increased vascularization and re-epithelialization. Taken together, these results clearly demonstrate that LL37 may have a key role in wound regeneration through vascularization.

The cathelicidin derived human peptide LL37 has a broad spectrum of antimicrobial and immunomodulatory activities. The large variety of biological activities makes LL37 a very promising candidate for clinical applications. The production of biologically active LL37 in large amounts with reduced costs can only be achieved using recombinant techniques. In this work, LL37 has been cloned to the N- and C-termini of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; both constructions (LL37-LK-CBM3 and LK-CBM3-LL37) were cloned into the pET-21a vector. A formic acid recognition site was introduced between the two modules, allowing the isolation of LL37 after chemical cleavage. The recombinant proteins were expressed in Escherichia coli BL21 (DE3) and solubilized with Triton X-100. The purification was achieved using cellulose CF11 fibers, taking advantage of the CBM3 specific affinity for cellulose; after hydrolysis with formic acid, LL37 was further purified by reverse-phase HPLC, as confirmed by MALDI-TOF mass spectrometry. The production and purification methodology developed in this work compares advantageously to other protocols previously described, having fewer purification steps. Only the recombinant LL37 obtained from the C-terminally fused protein (LK-CBM3-LL37) showed antibacterial activity against E. coli K12, with a MIC of 180 microg/ml.

Magainin-2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram-positive and gram-negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N-terminal of a family III carbohydrate-binding module fused to the linker sequence (LK-CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2-LK-CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK-CBM3. Its functionality was confirmed by testing its activity against gram-negative bacteria.