Avitabile, Angela (2017) Development of Antimicrobial peptides mixture with antibacterial and anti-inflammatory activity. [Tesi di dottorato]

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Abstract

Antimicrobial peptides (AMPs) are relatively short peptides endowed with broad spectrum antimicrobial, anti-inflammatory immunomodulating and wound healing enhancement activity. They are essential components of the innate immune system of higher eukaryotes providing the first line of defense against microbial invasions. AMPs are extremely heterogeneous in length, structure, aminoacid composition and, more importantly, molecular mechanism of bacterial killing. From this point of view AMPs can be divided in at least four wide classes: (i) cationic antimicrobial peptides (CAMPs); (ii) anionic antimicrobial peptides (AAMPs); (iii) histidine-rich antimicrobial peptides (HAMPs), and (iv) AMPs with intracellular targets (DNA, RNA, ribosomes, chaperons, enzymes). The pharmacological potential of AMPs, and especially of AMP mixtures, is obvious. However the costs of pharmaceutical formulations including two or more peptides separately produced would be so high that even a very interesting pharmacological activity would not compensate for the effort needed to prepare the mixture. Accordingly, the aim of the present project is to develop a strategy to prepare mixtures of two or more AMPs with costs, production times and amount of work comparable to those necessary for the production of a single recombinant AMP. Two alternative strategies were evaluated, one exploiting a previously optimized-carrier protein for the production of recombinant AMPs as fusion proteins, and an alternative strategy aiming to prepare a carrier-less precursor composed only by AMP modules. The carrier developed by our group, ONC-DCless-H6, was able to effectively drive to inclusion bodies both a highly cationic sequence of about 50 residues [(P)PAP-A3(Pro26)] containing two CAMP-like sequences and a modular sequence (AT2H) of more than 100 residues including 2 anionic, 2 cationic and a histidine rich module. In both cases expression yields were high (>200 mg/L and >100 mg/L, respectively). Purification by IMAC provided a protein with good yield and purity. Purified recombinant proteins were hydrolyzed in 0.1 M acetic acid at 60°C to cleave the acid labile Asp-Pro sequences at the junction sites between consecutive AMP modules. The efficiency was very high in the case AT2H, which provided only the desired peptides, thus indicating that all the acid labile Asp-Pro sequences underwent cleavage with an efficiency higher than 95%. Only in the case of (P)PAP-A3(Pro26), the internal Asp-Pro sequence was cleaved with an efficiency of only about 85%. The comparison of the sequences adjacent to the different Asp-Pro cleavage sites in (P)PAP-A3(Pro26) and AT2H suggested that in order to obtain a very high cleavage efficiency a flexible sequence, rich in small and/or coil-preferring amino acids should be present at least on one side of the Asp-Pro site. This information will be useful for the design of further fusion proteins. The carrier-less strategy was tested by preparing two modular precursors, AT5H (containing 5 anionic, 5 cationic and a histidine rich module) and AT4LH (containing 4 anionic, 4 cationic and a histidine rich module). AT5H and AT4LH showed relatively low expression levels (15 and 30 mg/L, respectively) and a different behavior of the inclusion bodies formed upon recombinant expression. In fact, the inclusion bodies, differently from those generated by ONC-DCless-H6, were soluble in 2 M urea thus suggesting a lower compactness/stability. A possible cause of the low expression levels could be the non-optimal codon usage of the coding sequences necessary for avoiding the presence of identical direct repeats which would have made difficult the chemical synthesis of the coding sequences. Low expression levels, in turn, could have contributed to the formation of “unstable” inclusion bodies. Indeed, it is well known that inclusion bodies formation is generally related to high expression levels. In the future, alternative approaches based on ligation of multiple copies of a smaller fragment could allow preparing coding sequences where all the repeats have an optimal codon usage.

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