Functional and structural evaluation of the antileukaemic enzymel-asparaginase II expressed at low temperature by differentEscherichia colistrains

Author(s)

de Moura, Werner Alfinito Feio ; Schultz, Leonardo ; Breyer, Carlos Alexandre ; de Oliveira, Ana Laura Pires ; Tairum, Carlos Abrunhosa ; Fernandes, Gabriella Costa ; Toyama, Marcos Hikari ; Pessoa-Jr, Adalberto ; Monteiro, Gisele ; de Oliveira, Marcos Antonio

• Document type: Journal article
• Source: Biotechnology Letters; v. 42, n. 11, p. 12-pg., 2020-07-07.

Abstract

Acute lymphoblastic leukaemia (ALL) affects lymphoblastic cells and is the most common neoplasm during childhood. Among the pharmaceuticals used in the treatment protocols for ALL, Asparaginase (ASNase) fromEscherichia coli(EcAII) is an essential biodrug. Meanwhile, the use of EcAII in neoplastic treatments causes several side effects, such as immunological reactions, hepatotoxicity, neurotoxicity, depression, and coagulation abnormalities. Commercial EcAII is expressed as a recombinant protein, similar to novel enzymes from different organisms; in fact, EcAII is a tetrameric enzyme with high molecular weight (140 kDa), and its overexpression in recombinant systems often results in bacterial cell death or the production of aggregated or inactive EcAII protein, which is related to the formation of inclusion bodies. On the other hand, several commercial expression strains have been developed to overcome these expression issues, but no studies on a systematic evaluation of theE. colistrains aiming to express recombinant asparaginases have been performed to date. In this study, we evaluated eleven expression strains at a low temperature (16 degrees C) with different characteristics to determine which is the most appropriate for asparaginase expression; recombinant wild-type EcAII (rEcAII) was used as a prototype enzyme and the secondary structure content, oligomeric state, aggregation and specific activity of the enzymes were assessed. Structural analysis suggested that a correctly folded tetrameric rEcAII was obtained using ArcticExpress (DE3), a strain that co-express chaperonins, while all other strains produced poorly folded proteins. Additionally, the enzymatic assays showed high specific activity of proteins expressed by ArcticExpress (DE3) when compared to the other strains used in this work. (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: 11/13500-6 - Investigation of the molecular determinants involved in the interaction with substrates of Tsa1 and Tsa2 of Saccharomyces cerevisiae
• Grantee: Carlos Alexandre Breyer
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 16/19245-1 - Evaluation of bacterial strains to L-asparaginase super expression: an antileukemic enzime from Escherichia coli
• Grantee: Werner Alfinito Feio de Moura
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 17/25272-4 - Asparaginase 1 from Saccharomyces cerevisiae: investigation of the functional and structural effects of the N-terminal domain
• Grantee: Gabriella Costa Fernandes
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• FAPESP's process: 19/04054-4 - Search of inhibitors of typical 2-Cys peroxirredoxin system from eukaryotes: functional and structural evaluation
• Grantee: Ana Laura Pires de Oliveira
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 17/19942-7 - Search for inhibitors of the peroxirredoxin system from pathogens and humans
• Grantee: Marcos Antonio de Oliveira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 14/22039-9 - Functional and structural characterization and rational modification of ASPaseM: a new pharmaceutical for the acute lymphoblastic leukemia treatment?
• Grantee: Leonardo Schultz da Silva
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 17/20291-0 - Characterization of the proinflammatory activity of a new serine protease (cdtsp-2) purified from the total venom of Crotalus durissus terrificus
• Grantee: Marcos Hikari Toyama
• Support Opportunities: Regular Research Grants