| Congresso Brasileiro de Microbiologia 2023 | Resumo: 852-1 | ||||
Resumo:L-asparaginase (L-ASNase) is a biopharmaceutical used for the treatment of Acute Lymphoid Leukemia (ALL). This drug promotes the hydrolysis of the bloodstream asparagine, leading to the death of leukemic cells since they are unable to synthesize asparagine. Although L-ASNase has been employed in clinical practice for decades, plenty of adverse reactions, such as allergies and anaphylactic shock, are frequently reported. These reactions are related to the bacterial origin of the formulations available. In this scenario, this work aims to explore fungi biodiversity, expecting to obtain an L-ASNase with less immunogenicity. Penicillium cerradense (GenBank: MT742156.1) L-ASNase gene was used in this study. A vector pET28-L-ASNasePC containing the P. cerradense L-ASNase optimized codon gene with a 6x-His Tag synthesis sequence in the N-terminal and a thrombin site for posterior tag excision was constructed. Competent E. coli BL21(DE3) cells were transformed with the vector. Ten clones randomly selected had their confirmation of transformation by Polymerase Chain Reaction (PCR) and were cultivated in Luria-Bertani (LB) medium, induced with 0,5 mM of Isopropil β-D-1 tiogalactoriranoside (IPTG), at 37 ºC, 200 rpm, during 18 h. After the extraction of proteins by sonication, L-ASNase expression was evaluated through SDS-PAGE 12% and Western Blotting (WB), using an anti-6x-His Tag antibody. One promising clone was selected and IPTG concentration, temperature, and pos-time induction were evaluated to obtain the best L-ASNase expression conditions. Finally, the protein extracts were submitted to SDS-PAGE 12% and L-ASNase activity was measured by Nesslerization. After expression conditions were optimized, a 200 mL culture was performed in Erlenmeyer 1 L, using the best conditions. The inclusion bodies (5 mg) were solubilized with urea 1-8 M, 20 mM β-mercaptoethanol and 1 mM phenylmethylsulfonylfluoride (PMSF) at phosphate saline buffer (PBS), pH 8.5. The solubilized inclusion bodies are purified for affinity chromatography, using nickel columm (HisTrapTMHP, Cytiva™). The purification were confirmed with SDS-PAGE 12%. The activity enzymatic was performed after desalting with PD-10 columm deslating (Cytiva™). After transformation, the selection and confirmation of the expression through WB, L-ASNase expression was evaluated in different temperatures, post-time expression, and IPTG concentration. There was no L-ASNase activity in any condition, even when it was expressed. This indicates the formation of inclusion bodies. After SDS-PAGE analysis, the best condition to performer fermentation was 37 ºC, 0,5 mM IPTG and 4 h after induction. After 200 mL culture performed, totality inclusion bodies were solubilized with buffer containing urea 7 M. SDS-PAGE indicated the L-ASNase was eluted. There was no L-ASNase activity after dessalting. Although the enzymatic activity was not detected, high levels of expression were confirmed by SDS-PAGE and WB. Refoldig techniques for recovering biologically active L-ASNase will be evaluate. Palavras-chave: Acute lymphoid leukemia, enzyme, fermentation processes, inclusion bodies, L-asparaginase Agência de fomento:Process number 193.0000919-2020/07. CHAMADA CONJUNTA FAPDF E FAPESP Nº 01/2019 and CAPES. | |||||