Carrera de Biotecnología
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Item Obtención de un péptido antimicrobiano autoescindible inducido por inteina utilizando un sistema de expresión en Escherichia coli(Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Biotecnología, 2023-03) Cordones Chimborazo, Kerly Lissette; García Solís, Mario DanielNowadays, there is a high prevalence of antibiotic resistance, which poses a danger to public health. In response to this global problem, arises the creation of antimicrobial peptides (PAMs), due to the wide antibacterial spectrum they exhibit. Based on this, cecropins sequences were used to design a peptide through ancestral reconstruction, from which an ancestral peptide called CRP_N0 was obtained. The current methods for the production of PAMs are chemical synthesis, plant cell production and direct extraction from natural sources. However, these methods have a high economic value and a low efficiency. Therefore, the peptide CRP_N0 was expressed by an intein-induced self-splitting system linked by its N-terminal end to six histidines. The six-histidine label was used to purify the fusion protein intein-CRP_N0 and the removal of intein from the peptide CRP_N0. The construction was inserted into the plasmid pET-11b and expressed in Escherichia coli BL21 pG-Tf1. To maximize the expression of the fission protein, different induction times were tested with IPTG and the optimal expression time was determined to be 4 hours. In addition, an excision test was also conducted in which different temperature and pH conditions were evaluated to induce self-excision of inteine, where it was determined that at 25 degrees Celsius and a basic pH (10) a high yield is obtained in the cutting of intein.