Production of a novel N-terminal PEGylated crisantaspase

Autor(es)

Torres-Obreque, Karin ^[1] ; Meneguetti, Giovanna Pastore ^[1] ; Custodio, Debora ^[1] ; Monteiro, Gisele ^[1] ; Pessoa-Junior, Adalberto ^[1] ; de Oliveira Rangel-Yagui, Carlota ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, Dept Biochem & Pharmaceut Technol, Ave Prof Lineu Prestes, 580 Bl 16 Butanta, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Biotechnology and Applied Biochemistry; v. 66, n. 3, p. 281-289, MAY-JUN 2019.
• Citações Web of Science: 1

Resumo

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L-1 H-1, respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg(-1), 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a K-M value three times higher than crisantaspase (150 and 48.5 mu M, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL. (AU)

• Processo FAPESP: 16/22065-5 - Pegilação N-terminal de proteínas e purificação por sistemas aquosos bifásicos
• Beneficiário: Carlota de Oliveira Rangel Yagui
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• 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