Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host

Autor(es)

Santos, Joao H. P. M. ; Costa, Iris M. ; Molino, Joao V. D. ; Leite, Mariana S. M. ; Pimenta, Marcela V. ; Coutinho, Joao A. P. ; Pessoa, Jr., Adalberto ; Ventura, Sonia P. M. ; Lopes, Andre M. ; Monteiro, Gisele

• Tipo de documento: Artigo Científico
• Fonte: BIOTECHNOLOGY PROGRESS; v. 33, n. 2, p. 416-424, MAR-APR 2017.
• Citações Web of Science: 3

Resumo

l-asparaginase (ASNase) is a biopharmaceutical widely used to treat child leukemia. However, it presents some side effects, and in order to provide an alternative biopharmaceutical, in this work, the genes encoding ASNase from Saccharomyces cerevisiae (Sc\_ASNaseI and Sc\_ASNaseII) were cloned in the prokaryotic expression system Escherichia coli. In the 93 different expression conditions tested, the Sc\_ASNaseII protein was always obtained as an insoluble and inactive form. However, the Sc\_ASNaseI (His)(6)-tagged recombinant protein was produced in large amounts in the soluble fraction of the protein extract. Affinity chromatography was performed on a Fast Protein Liquid Chromatography (FPLC) system using Ni2+-charged, HiTrap Immobilized Metal ion Affinity Chromatography (IMAC) FF in order to purify active Sc\_ASNaseI recombinant protein. The results suggest that the strategy for the expression and purification of this potential new biopharmaceutical protein with lower side effects was efficient since high amounts of soluble Sc\_ASNaseI with high specific activity (110.1 +/- 0.3 IUmg(-1)) were obtained. In addition, the use of FPLC-IMAC proved to be an efficient tool in the purification of this enzyme, since a good recovery (40.50 +/- 0.01%) was achieved with a purification factor of 17-fold. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017 (AU)

• Processo FAPESP: 15/07749-2 - Engenharia de proteínas e comparação de sistemas microbianos de expressão do biofármaco L-asparaginase
• Beneficiário: Gisele Monteiro
• Linha de fomento: 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
• Linha de fomento: Auxílio à Pesquisa - Temático