Abstract
Antimicrobial peptides (AMPs) are promising candidates for new generations of antibiotics to overcome the threats of multidrug-resistant infections as well as other industrial applications. Recombinant expression of small peptides is challenging due to low expression rates and high sensitivity to proteases. However, recombinant multimeric or fusion expression of AMPs facilitates cost-effective large-scale production of AMPs. In This project, S3 and SΔ3 AMPs were expressed as fusion partners. S3 peptide is a 34 amino acid linear antimicrobial peptide derived from lipopolysaccharide (LPS) binding site of factor C of horseshoe crab hemolymph and SΔ3 is a modified variant of S3 possessing more positive charges. Two copy tandem repeat of the fusion protein (named as SΔ3S3-2mer-GS using glycine- serine linker was expressed in E. coli. BL21 (DE3). After cell disruption and solubilization of inclusion bodies, the protein was purified by Ni -NTA affinity chromatography. Antimicrobial activity and cytotoxic properties of purified SΔ3S3-2mer-GS were compared with a previously produced tetramer of S3 with the same glycine- serine linker (S3-4mer-GS) and each of monomeric blocks of S3 and SΔ3. SΔ3S3-2mer-GS was successfully expressed with an expression rate of 26%. The geometric average of minimum inhibitory concentration (MIC GM) of SΔ3S3-2mer-GS was 28%, 34%, and 57% lower than SΔ3, S3-4mer-GS, and S3, respectively. SΔ3S3-2mer-GS had no toxic effect on eukaryotes human embryonic kidney cells at its MIC concentration. tandem repeated fusion expression strategy could be employed as an effective technique for recombinant production of AMPs.
Highlights
Microbial resistance to antibiotics has been reported annually even monthly with a gradient increasing which made a need for approaching more effective antimicrobial treatments [1, 2]
Protein expression and purification The designed plasmid was amplified at E. coli Top 10 and after extraction and purification, was transferred to E. coli BL21 (DE3) strain
S3 peptide was expressed as a tetrameric form in E. coli with two different aspartic acid-proline (S34mer-DP) and glycine-serine linker (S3-4merGS)
Summary
Microbial resistance to antibiotics has been reported annually even monthly with a gradient increasing which made a need for approaching more effective antimicrobial treatments [1, 2]. Increasing net positive charges of AMPs by adding positively charged (lysine or arginine) residues[5,6,7,8,9], expelling or replacing negatively charged (glutamic acid or aspartic acid) residues[10], increasing amphipathicity by adding hydrophobic residues [11,12,13,14,15,16] or AMPs hybridization [17] may enhance antimicrobial activity and/or lipopolysaccharide (LPS) binding affinity of AMPs without considerable intensification of their cytotoxicity. Methods: Two copy tandem repeat of the fusion protein S∆3S3-2mer-GS had no toxic effect on eukaryotes human embryonic kidney cells at its MIC concentration. Conclusions: tandem repeated fusion expression strategy could be employed as an effective technique for recombinant production of AMPs
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