| Congresso Brasileiro de Microbiologia 2023 | Resumo: 686-2 | ||||
Resumo:TITLE: A chimeric protein engineered of L-asparaginase and Arginase I for the treatment of leukemia: production, in silico analysis and enzymatic evaluation.
AUTHORS: RIVERA, M.; MONTEIRO, G.
INSTITUTION: Universidade de São Paulo, São Paulo, SP (Av. Prof. Lineu Prestes, 580 - Butantã, São Paulo).
ABSTRACT:
The enzymatic biopharmaceutical L-asparaginase has been used for more than 40 years as a second-line treatment for acute lymphoblastic leukemia (ALL). Although its use has provided high remission rates, neurological and immunogenic side effects have been reported, the latter due to its bacterial origin. That is why it is necessary to look for new sources of the enzyme or to use protein engineering techniques to improve the molecule in order to mitigate said effects, in addition to improving its anticancer activity. This study reports the construction and evaluation of a bifunctional complex of L-asparaginase enzymes (clone 63N-hC consisting of gpASNase1 and hASNase1 L-asparaginases) fused to the N-terminus of the enzyme human Arginase I (ARG1) and joined by a rigid peptide linker. The in silico analysis of the three-dimensional structure of the protein monomer suggested a high quality of the model in relation to the position of each enzyme in the construct (N,C-terminal) and in relation to their positions relative to the domains that contain the catalytic centers. The molecular docking results show that the rigid linker allows the independent folding of each enzyme, which generates stability, in addition to good coupling with the respective substrates. Different protein expression tests were developed with the recombinant vector 63N-hC -linker-ARG1/pET-22b(+) and with Escherichia coli strains ArcticExpress (DE3), BL21 (DE3) and AD494. The tests were successful when observing the expression of the chimeric protein for the three strains in cultivation conditions of 0.01 mM of the IPTG inducer (Isopropyl-β-D-1-thiogalactopyranoside), 16 hours and 20°C of temperature post-induction. The protein was expressed in the insoluble fraction in the form of inclusion bodies, and even in this state, both enzymes exhibited the parental enzymatic activities indicating the presence of non-classical inclusion bodies. Having noted that the highest enzymatic activity values were achieved for the protein expressed in the ArcticExpress strain (DE3), with values of 2.69 ± 0.8 U/mL for L-asparaginase activity and 13.56 ± 1.6 U/mL for L for Arginase activity. For the management of the inclusion bodies, a classic extraction methodology was used, which consists of a combined method of sonication and enzymatic lysis, followed by a solubilization step with urea or sodium dodecyl sulfate, and finally a refolding protein step using buffers with additives that promote protein refolding and breakdown such as arginine and glycerol. The highest total protein concentration, indicating the highest amount of extracted inclusion bodies, was obtained in the ArcticExpress (DE3) strain cultures (52 ± 4.0 mg/mL). However, the recovery of enzymatic activities for both enzymes was not so successful, possibly indicating that the method used to refold was not as effective, even so, the results presented in this study point to the viability and functionality of the constructed bifunctional protein complex.
Keywords: Arginase I, chimeric protein, inclusion bodies, L-asparaginase.
Development Agency: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES.
Palavras-chave: Arginase I, chimeric protein, inclusion bodies, L-asparaginase Agência de fomento:Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES | |||||