Efeito citotóxico e imunogenicidade in silico de L-asparaginase recombinante de Phaseolus vulgaris L.

• Ano de defesa: 2023
• Autor(a) principal: Gomes, José Gabriel da Silva
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Enzima anticancerígena; Feijão; Proteína recombinante; Citotoxicidade
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/74046

The enzyme L-asparaginase plays a crucial role in the treatment of acute lymphoblastic leukemia, a type of cancer that mostly affects children and teenagers. However, due to their immunogenicity and glutaminase activity, it is common for these molecules to cause adverse reactions during treatment. These downsides ignite the search for novel asparaginases that could mitigate these problems. In this quest, plants have shown promising features over the years that turn them into candidates to substitute the commercial enzymes, which are from bacterial source. In this context, Phaseolus vulgaris recombinant L-asparaginase lacks a thorough characterization of its biochemical, biophysical and antineoplastic properties. Thus, this work aimed to produce and characterize a recombinant L-asparaginase from P. vulgaris (Asp-P). For this purpose, the enzyme was expressed in E. coli and purified by affinity and size-exclusion chromatographies. The enzyme activity was measured by the Nesslerization method. The kinetics parameters, thermotolerance and the effects of interferents on enzyme activity were determined. The presence of the enzyme in the samples was analyzed by western blotting and mass spectrometry. The secondary structure and the thermostability of the protein were both assessed by circular dichroism. Also, the cytotoxicity effects of Asp-P on Raji and K562 cells were assayed by MTT method. Online prediction tools were used to determine the immunogenicity of Asp-P in comparison with the bacterial protein. The results showed that Asp-P was expressed with high yields and specific activity of 905 U.mg-1 , with maximum activity at pH 9.0 and 40° C. Western blots confirmed the presence of Asp-P in the samples, as well as no contaminant native E. coli L-asparaginase. Mass spectrometry mapped 93 % Asp-P sequence. Asp-P could reduce Raji viable cells percentage below 50 % only at the highest concentration tested, but the same could not be achieved for K562 cultures. Lastly, in silico prediction methods indicated that Asp-P is less immunogenic than bacterial enzymes. Put together, these results show that recombinant Asp-P has different properties when compared to the native enzyme and has favorable aspects that indicate a promising enzyme to substitute the bacterial ones.