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

Author(s)

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

• Document type: Journal article
• Source: PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY; v. N/A, p. 12-pg., 2022-08-16.

Abstract

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)

• FAPESP's process: 13/08617-7 - Production of extracellular L-asparaginase: from bioprospecting to the engineering of an antileukemic biopharmaceutical
• Grantee: Adalberto Pessoa Junior
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/15104-0 - Preclinical assays of glycoprotein asparaginase proteoforms or resistant to serum proteases.
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants
• FAPESP's process: 16/09138-3 - Engineering of the signal secretory portion of asparaginase from Saccharomyces cerevisiae for extracellular biopharmaceutical production
• Grantee: Henrique Pellin Biasoto
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 15/07749-2 - Protein engineering and comparison of microbial expression systems of the biopharmaceutical L-asparaginase
• Grantee: Gisele Monteiro
• Support Opportunities: Regular Research Grants