Saccharomyces cerevisiae asparaginase II, a potential antileukemic drug: Purification and characterization of the enzyme expressed in Pichia pastoris.

Autor: Facchinetti de Castro Girão L; Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil; Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil., Gonçalves da Rocha SL; Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil., Sobral RS; Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil., Dinis Ano Bom AP; Oswaldo Cruz Foundation, Biomanguinhos, Laboratory of Macromolecules, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil., Franco Sampaio AL; Oswaldo Cruz Foundation, Farmanguinhos, Institute of Pharmaceutical Technology, 21041-250, Rio de Janeiro, Rio de Janeiro, Brazil., Godinho da Silva J Jr; Oswaldo Cruz Foundation, Biomanguinhos, Laboratory of Macromolecules, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil., Ferrara MA; Oswaldo Cruz Foundation, Farmanguinhos, Drugs Technology Laboratory, 21041-250, Rio de Janeiro, Rio de Janeiro, Brazil., Pinto da Silva Bon E; Federal University of Rio de Janeiro, Institute of Chemistry, Department of Biochemistry, Enzyme Technology Laboratory, 21941-909, Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: elba1996@iq.ufrj.br., Perales J; Oswaldo Cruz Foundation, Oswaldo Cruz Institute, Toxinology Laboratory, 21040-900, Rio de Janeiro, Rio de Janeiro, Brazil. Electronic address: jperales@ioc.fiocruz.br.
Jazyk: angličtina
Zdroj: Protein expression and purification [Protein Expr Purif] 2016 Apr; Vol. 120, pp. 118-25. Date of Electronic Publication: 2015 Dec 20.
DOI: 10.1016/j.pep.2015.12.012
Abstrakt: Asparaginase obtained from Escherichia coli and Erwinia chrysanthemi are used to treat acute lymphocytic leukaemia and non-Hodgkin's lymphoma. However, these agents cause severe adverse effects. Saccharomyces cerevisiae asparaginase II, encoded by the ASP3 gene, could be a potential candidate for the formulation of new drugs. This work aimed to purify and characterize the periplasmic asparaginase produced by a recombinant Pichia pastoris strain harbouring the ASP3 gene. The enzyme was purified to homogeneity with an activity recovery of 51.3%. The estimated molecular mass of the enzyme was 136 kDa (under native conditions) and 48.6 kDa and 44.6 kDa (under reducing conditions), suggesting an oligomeric structure. The recombinant asparaginase is apparently non-phosphorylated, and the major difference between the monomers seems to be their degree of glycosylation. The enzyme showed an isoelectric point of 4.5 and maximum activity at 46 °C and pH 7.2, retaining 92% of the activity at 37 °C. Circular dichroism and fluorescence analyses showed that the enzyme structure is predominantly α-helical with the contribution of β-sheet and that it remains stable up to 45 °C and in the pH range of 6-10. In vitro tests indicated that the recombinant asparaginase demonstrated antitumoural activity against K562 leukaemic cells.
(Copyright © 2015 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE
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