Antimicrobial peptides are natural substances that have played a role in the development of the adaptive immune system, and are currently involved in the prevention of infections, through their direct antimicrobial and immunomodulatory properties. While the amino acid composition and spatial structure vary, most antibacterial peptides have a positive surface charge, which allows them to bind to the negative bacterial membranes. Buforin II is a widely studied antimicrobial peptide first obtained through the structural modification of buforin I, a peptide isolated from Bufo gargarizans. The peptide showed significant antibacterial activity against Gram-positive and Gram-negative bacterial strains. The mechanism of action of buforin II differs from that of other antimicrobial peptides, as it binds directly to bacterial DNA and RNA. The aim of our study was to obtain recombinant buforin II with a ubiquitin fusion partner, through heterologous expression in Escherichia coli Rosetta™ (DE3)pLysS cells, using a laboratory scale bioreactor. The incubation of expression host cells in a bioreactor allowed the constant monitoring and control of the process parameters, leading to high biomass levels and an increased production rate of the peptide. The parameters used during incubation were: 37°C, pH=6.9 and dissolved oxygen level above 40%. Purification of the recombinant protein was accomplished by affinity chromatography using a Ni-chelate solid phase to which the 10xHistag of our construct showed affinity. Method optimisation consisted in the use of gradient and linear elution, of which the latter was found to be more effective. Digestion of the fusion partner from the target peptide was performed with ubiquitin carboxyl-terminal hydrolase enzyme. The expression and purification protocols developed in our experiment allow the production of a significant amount of buforin II, allowing its use for further research. Furthermore, the presented methods could be suitable for industrial production of the recombinant peptide..

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Life Sciences, other, Medicine, Clinical Medicine, Pharmacy