Effect of Flexible and Rigid Linkers on Biological Activity of Recombinant Tetramer Variants of S3 Antimicrobial Peptide

Antimicrobial peptides (AMPs) mainly introduced as a new generation of antibiotics, could be used for broad medical and biotechnological applications including treatment of multi drug resistant bacterial infections, anti-biofilm agents, wound healing, drug delivery and biosensors. High cost of peptide synthesis is one of the major obstacles of industrial scale production of AMPs. Recombinant expression of tandem repeats (multimeric forms) of AMPs with proper linkers is a useful approach for overcoming the high cost of peptides synthesis. In the present study, the effect of using glycine- serine [(GGGGS) 3] and aspartic acid-proline (DP) Linkers between four tandem repeats of sushi S3 antimicrobial peptide on its biological activity was investigated. S3 peptide, derived from factor C of horseshoe crab hemolymph has been known as a promising candidate as anti-Gram negative bacterial agent. Each variant of S3 tetramers with (GGGGS) 3 and DP linkers (named S3-4mer-GS and S3-4mer-DP, respectively) were expressed in E. coli. BL21 (DE3) as inclusion bodies and were purified by using immobilized metal affinity chromatography (IMAC). Antimicrobial activities and cytotoxic properties of variants were evaluated by determination of minimum inhibitory concentration assay (MIC) and MTT assay. Statistical analysis revealed that glycine serine linker improved antimicrobial activity of S3-tetramer approximately 25% and 86% in comparison to tetramer with aspartic acid - proline linker and S3 monomer respectively. According to the enhanced antibacterial activity of glycine- serine linker without any significant effects on its cytotoxicity, this linker will be a good choice for recombinant tandem expression of small antimicrobial peptides.