Produção do peptídeo antimicrobiano BotrAMP14 de interesse na pecuária para mastite bovina

• Ano de defesa: 2023
• Autor(a) principal: Lima, Letícia Ferreira
• Orientador(a): Franco, Octávio Luiz
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Católica de Brasília
• Programa de Pós-Graduação: Programa Stricto Sensu em Ciências Genômicas e Biotecnologia
• Departamento: Escola de Saúde e Medicina
• País: Brasil
• Palavras-chave em Português: Mastite bovina; Peptídeo antimicrobiano; Bioprocessos; Polypeptides; Inteína
• Palavras-chave em Inglês: Bovine mastitis; Antimicrobial peptides; Bioprocess; Elastin-like; Intein
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS
• Link de acesso: https://bdtd.ucb.br:8443/jspui/handle/tede/3175

Livestock farming is extremely important to the Brazilian economy. Nevertheless, bovine mastitis infections can negatively impact this sector. Currently, the treatment occurs throughout antibiotics usage, but their inappropriate utilization can generate microbial resistance and/or residues in animals. In that view, antimicrobial peptides are an interesting alternative against bovine mastitis, since such molecules demonstrate activity against several pathogens. In the previous group study, BotrAMP14 peptide didn’t demonstrate cytotoxic in RAW cells, even at 128 µM concentration. In vivo assays showed BotrAMP14 activity at 64 µM in mice against bovine mastitis microorganisms. This work aims to perform BotrAMP14 heterologous expression associated with ELP-intein, using the host system bacterium Escherichia coli. Thereby, data here reported that BotrAMP14 peptide associated with ELP-intein was successfully expressed. The SDS-PAGE analysis showed the expected molecular mass of 47 kDa and the 1988 ion corresponding to the hybrid protein fragment was found in the MALDI-ToF analysis. Bioreactor assays demonstrated that lactose 10 g.L 1 caused metabolic stress in E. coli cells resulting in metabolic deviation. Subsequently, 2.05 mg.mL-1 of partially purified hybrid protein was obtained from hysteresis purification. Molecular modeling and docking suggest high affinity to intein-BotrAMP14 interaction of -5.5 kcal.mol-1 due hydrogen bonds, electrostatic interactions, and hydrophobic interactions. However, it was not possible to activate the intein for peptide release. In this way, it is possible to infer based on in silico that BotrAMP14 does not release from the complex due to intein protein ligation. In this view, this study evidences the heterologous expression as low-cost production aiming their utilization for bovine mastitis treatment.