Tesis Doctorado

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Browsing Tesis Doctorado by Author "Aguilar Domínguez, Pedro Pablo"

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    Desarrollo y producción recombinante de un péptido bloqueador de TNF para el tratamiento de trastornos autoinmunes inflamatorios.
    (Universidad de Concepción, 2023) Aguilar Domínguez, Pedro Pablo; Sánchez Ramos, Oliberto; Zaror Zaror, Claudio
    Tumor Necrosis Factor has become an important therapeutic target for the treatment of inflammatory autoimmune disorders. To date, there are five FDA-approved TNF-blocking biopharmaceuticals that correspond to fusion proteins and monoclonal antibodies. Despite the reported therapeutic results, these biopharmaceuticals have drawbacks that limit their use and a high production cost in mammalian cells. In this study, we designed a recombinant TNF blocking peptide (CBB288). Our approach consisted in a recombinant tandem protein containing 15 copies of the peptide was obtained in E. coli. The tandem protein expression was induced using IPTG. The hydrolysis of the tandem protein to obtain monomeric units of the CBB288 peptide was performed using the hydroxylamine method. Recombinant peptide (r-CBB288) exhibited a high binding affinity to TNF with KD=2,39x10-5 ± 0,15 mol.L-1 , similarly to synthetic homologue peptide (syn-CBB288); KD=1,03x10-5 ± 0,05 mol.L-1 . The TNF-neutralizing activity of r CBB288 was assessed in L929 cells. r-CBB288 was able to inhibit TNF induced cytotoxicity in a dose response manner, resulting in a maximum viability recovery of 79%. According to an in silico simulation study, it was demonstrated that the differences between the synthetic and recombinant peptides do not modify the predicted binding affinity for the target protein. The results obtained suggest that CBB288 could be obtained by a recombinant process and used for future biomedical studies related to chronic inflammation pathologies.
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