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
palavras-chave
ACUTE LYMPHOBLASTIC-LEUKEMIA; RECOMBINANT PROTEIN EXPRESSION; GLUTAMINASE ACTIVITIES; PANCREATITIS; CHILDREN; CHROMATOGRAPHY; THERAPY
categoria
Biotechnology & Applied Microbiology; Food Science & Technology
autores
Santos, JHPM; Costa, IM; Molino, JVD; Leite, MSM; Pimenta, MV; Coutinho, JAP; Pessoa, A; Ventura, SPM; Lopes, AM; Monteiro, G
nossos autores
João Henrique Picado Madalena Santos
Estudante de doutoramento
Sónia Patrícia Marques Ventura
Professor AuxiliarProjectos
CICECO - Aveiro Institute of Materials (UID/CTM/50011/2013)
Projeto de Investigação Exploratória: Sónia Patricia Marques Ventura (IF/00402/2015)
agradecimentos
This research was supported by grants from the Coordination for Higher Level Graduate Improvements (CAPES/Brazil), National Council for Scientific and Technological Development (CNPq/Brazil) and State of Sao Paulo Research Foundation (FAPESP/Brazil, process numbers 2013/08617-7 and 2015/07749-2). This work was developed in the scope of the project CICECO-Aveiro Institute of Materials (Ref. FCT UID/CTM/50011/2013), financed by national funds through the FCT/MEC. The authors also thank FCT for the doctoral SFRH/BD/102915/2014 and post-doctoral grants SFRH/BPD/79263/2011 of J.H.P.M. Santos and S.P.M. Ventura, respectively. JS doctoral grant and when applicable co-financed by FEDER under the PT2020 Partnership Agreement. Moreover, S.P.M. Ventura acknowledges the FCT/MEC (Portugal) for a contract under Investigador FCT 2015 contract number IF/00402/2015.