Heterologous expression and purification of active L-asparaginase I of Saccharomyces cerevisiae in Escherichia coli host
authors Santos, JHPM; Costa, IM; Molino, JVD; Leite, MSM; Pimenta, MV; Coutinho, JAP; Pessoa, A; Ventura, SPM; Lopes, AM; Monteiro, G
nationality International
journal BIOTECHNOLOGY PROGRESS
author keywords biopharmaceutical; fast protein liquid chromatography; protein purification; recombinant DNA; enzyme technology
keywords ACUTE LYMPHOBLASTIC-LEUKEMIA; RECOMBINANT PROTEIN EXPRESSION; GLUTAMINASE ACTIVITIES; PANCREATITIS; CHILDREN; CHROMATOGRAPHY; THERAPY
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 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
publisher WILEY
issn 8756-7938
year published 2017
volume 33
issue 2
beginning page 416
ending page 424
digital object identifier (doi) 10.1002/btpr.2410
web of science category Biotechnology & Applied Microbiology; Food Science & Technology
subject category Biotechnology & Applied Microbiology; Food Science & Technology
unique article identifier WOS:000400015900016
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  impact metrics
times cited (wos core): 0
journal impact factor (jcr 2016): 1.986
5 year journal impact factor (jcr 2016): 1.975
category normalized journal impact factor percentile (jcr 2016): 56.399
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