EVALUATION OF ANTIMICROBIAL ACTIVITY OF RECOMBINANT ENGINEERED SMALL PEPTIDES EXPRESSED IN PICHIA PASTORISGOLLA KAMALA, SESHA SRINIVAS VUTUKURU, JAYASREE GANUGAPATI, J. USHA RANI AND CHAND PASHAAbstract Antimicrobial peptides are the alternatives to antibiotics to reduce the drug resistance. In this pursuit, mechanism and empiricism-based protein engineering of LL-37, Buforin II and Pexiganan antimicrobial peptides was carried out and resulted in 200 variable peptides. Subsequently, 3D models were developed in Swiss model and PEPFOLD server. Highly stable 20 each LL-37, Buforin II and Pexigananvariants were docked (Cluspro 2.0 server) with FGFR/FGF which promotes angiogenesis and proliferation in higher mammals. The 200th LL-37, 192th Buforin II and 120thpexigananmodels showed relatively less interactions compared with wild peptides. A part from these three engineered peptides seven wild peptides and one cell penetrating peptide were connected with hydroxylamine digestion site. The wild and engineered peptides fusion was expressed in methylotrophic yeast, Pichia pastoris. The 777 bps gene fragment of fused AMPs was cloned into vector pPICZáA, transformed into Pichia pastoris X-33 for extracellular expression. The expressed recombinant peptide was purified by Ni-agarose affinity. Fused protein was digested with hydroxylamine and separated by size exclusion chromatography. Purified peptides and supernatant were used for antimicrobial assay against four pathogens, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Pseudomonas aeruginosa by well diffusion and rideal walker test. The results revealed that the three engineered and wild AMPs cocktail demonstrated good inhibitory effect against four tested bacteria and MIC was found to be 0.25 to 0.5μg/mL.
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