title: Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host.
abstract: 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 IU mg-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. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:416-424, 2017.
authors.affiliation: Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, 3810-193, Portugal.|Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: João H P M
authors.initials: JHPM
authors.lastname: Santos
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: Iris M
authors.initials: IM
authors.lastname: Costa
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: João V D
authors.initials: JVD
authors.lastname: Molino
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: Mariana S M
authors.initials: MSM
authors.lastname: Leite
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: Marcela V
authors.initials: MV
authors.lastname: Pimenta
authors.affiliation: Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, 3810-193, Portugal.
authors.forename: João A P
authors.initials: JAP
authors.lastname: Coutinho
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: Adalberto
authors.initials: A
authors.lastname: Pessoa
authors.affiliation: Department of Chemistry, CICECO-Aveiro Institute of Materials, University of Aveiro, Aveiro, 3810-193, Portugal.
authors.forename: Sónia P M
authors.initials: SPM
authors.lastname: Ventura
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: André M
authors.initials: AM
authors.lastname: Lopes
authors.affiliation: Department of Biochemical and Pharmaceutical Technology, School of Pharmaceutical Sciences, University of São Paulo-FCF/USP, São Paulo/SP, Brazil.
authors.forename: Gisele
authors.initials: G
authors.lastname: Monteiro
mesh_terms: D001215:Asparaginase / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; D001215:Asparaginase
keywords: biopharmaceutical; enzyme technology; fast protein liquid chromatography; protein purification; recombinant DNA