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Comparison between Two Erwinia carotovora L-Asparaginase II Constructions: cloning, Heterologous Expression, Purification, and Kinetic Characterization

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Author(s): Priscila Lamb Wink | Heique Marlis Bogdawa | Gaby Renard | Jocelei Maria Chies | Luiz Augusto Basso

Journal: Journal of Microbial & Biochemical Technology
ISSN 1948-5948

Volume: 02;
Issue: 01;
Start page: 013;
Date: 2010;
Original page

Keywords: L-asparaginase II | Erwinia carotovora | Heterologous expression | Cancer therapy | Therapeutic protein | Biosimilar

ABSTRACT
L-Asparaginase II from Erwinia carotovora may representan important alternative therapy in the treatment ofacute childhood lymphoblastic leukaemia, despite its promisinglower glutaminase activity than Escherichia coli andErwinia chrysanthemi L-asparaginases II, currently usedin treatment of this disease. Here we describe cloning, expression,purification and determination of steady-state kineticparameters for E. carotovora L-asparaginase II: with(AspSP) and without the signal peptide (AspMP). AspMPwas purified to homogeneity by a single-step protocol with91% yield, and AspSP by a two-step protocol with 28%yield. In addition, both enzymes presented similar high specificactivities: 208.1 and 237.6 U mg-1, respectively. TheKm and kcat values showed that AspMP has lower glutaminaseactivity than AspSP. Moreover AspMP is produced bya simpler purification protocol, and at higher yield. Thisprocess can be amenable to large scale production and beof interest to researchers and biopharmaceutical companies.
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