Development of a cell-free protein synthesis protocol to rapidly screen L-asparaginase proteoforms by enzymatic activity

Autor(es)

Lima, Guilherme M. ^[1] ; Menezes, Milene C. ^[2] ; Costa, Iris M. ^[1] ; Serrano, Solange M. T. ^[2] ; Monteiro, Gisele ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut, Dept Tecnol Bioquim Farmaceut, BR-05508000 Sao Paulo, SP - Brazil

^[2] Inst Butantan, Lab Toxinol Aplicada, Ctr Toxins Immune Response & Cell Signaling CeTIC, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF CHEMICAL TECHNOLOGY AND BIOTECHNOLOGY; v. 96, n. 9, p. 2659-2666, SEP 2021.
• Citações Web of Science: 0

Resumo

BACKGROUND Cell-free protein synthesis (CFPS) technology has emerged as a powerful tool for a variety of biotechnological applications, including the expression of different classes of biopharmaceutical products. L-Asparaginase (E.C. Number: 3.5.1.1, L-asparagine amidohydrolase) (L-ASNase) is an important biopharmaceutical used to treat leukemia, but expression of multiple proteoforms in CFPS systems and rapid characterization using standard colorimetric methods has not yet been fully exploited. Herein, recombinant expression and characterization of an L-ASNase from Erwinia chrysanthemi (Erwinase) using a new CFPS protocol is reported. RESULTS Expression and quantification of the enzymatic activity of a soluble his-tagged L-ASNase directly from a CFPS reaction was successfully achieved. Purification of the protein was not required in order to assess its biological activity. Activity of L-ASNase was significantly higher than the control reaction (7.07 +/- 0.68 U mL(-1) vs. 1.83 +/- 0.14 U mL(-1), respectively). Expression of a mutant Erwinase proteoform - V293M - was also achieved and it presented a similar enzymatic activity. No significant loss in L-ASNase enzymatic activity was noticed after removal of cyclic AMP, spermidine, transfer RNA, T7 RNA polymerase and, especially, ammonium acetate (a common interference in ASNase enzymatic assays) from the CFPS reaction. CONCLUSION The protocol developed in this work will facilitate the screening of novel clinically-relevant L-ASNase proteoforms. (c) 2021 Society of Chemical Industry (SCI). (AU)

• Processo FAPESP: 18/15041-8 - Expressão de L-asparaginase de Erwinia chrysanthemi em tecnologia de síntese proteica livre de células
• Beneficiário: Guilherme Meira Lima
• Modalidade de apoio: Bolsas no Brasil - Mestrado
• 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: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• 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: 16/25896-5 - Caracterização bioquímica e avaliação citotóxica de isoformas mutantes de L-Asparaginase II de Dickeya chrysanthemi (Erwinia chrysanthemi)
• Beneficiário: Iris Munhoz Costa
• Modalidade de apoio: Bolsas no Brasil - Doutorado