Extracellular expression of Saccharomyces cerevisiae's L-asparaginase II in Pichia pastoris results in novel enzyme with better parameters

Autor(es)

Biasoto, Henrique P. ; Hebeda, Cristina B. ; Farsky, Sandra H. P. ; Pessoa, Adalberto ; Costa-Silva, Tales A. ; Monteiro, Gisele

• Tipo de documento: Artigo Científico
• Fonte: PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY; v. N/A, p. 12-pg., 2022-08-16.

Resumo

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae's L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26-362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch (R) strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg(-1). The resultant enzyme is 40% more stable than commercially available Escherichia coli's ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi. (AU)

• Processo FAPESP: 13/08617-7 - Produção de L-asparaginase extracelular: da bioprospecção à engenharia de um biofármaco antileucêmico
• Beneficiário: Adalberto Pessoa Junior
• Modalidade de apoio: Auxílio à Pesquisa - Temático
• Processo FAPESP: 18/15104-0 - Ensaios pré-clínicos de proteoformas de asparaginase glicoproteicas ou resistentes a proteases séricas.
• Beneficiário: Gisele Monteiro
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 16/09138-3 - Engenharia da porção secretória sinalizadora de asparaginase de Saccharomyces cerevisiae para produção de biofármaco extracelular
• Beneficiário: Henrique Pellin Biasoto
• Modalidade de apoio: Bolsas no Brasil - Iniciação Científica
• Processo FAPESP: 15/07749-2 - Engenharia de proteínas e comparação de sistemas microbianos de expressão do biofármaco L-asparaginase
• Beneficiário: Gisele Monteiro
• Modalidade de apoio: Auxílio à Pesquisa - Regular