Cloning, expression, purification and characterisation of Erwinia carotovora L-asparaginase in Escherichia coli

Authors
Meraj Pourhossein
Hassan Korbekandi
Department of Anatomical Sciences and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
Abstract
Background: For the past 30 years, bacterial L-asparaginases have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. It is found in a variety of organisms such as microbes, plants and mammals. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. Our aim was to clone, express, purify and characterize E. carotovora asparaginase.
Materials and Methods: L-asparaginase from E. carotovora NCYC 1526 (ErA) was cloned and expressed in Escherichia coli strain BL21 (DE3). The enzyme was purified to homogeneity by affinity chromatography. Various conditions were tested to maximize the production of recombinant asparaginase in E. coli.
Results: A new L. asparaginase from E. carotovora NCYC 1526 (ErA) was successfully cloned, expressed and purified in E. coli BL21 (DE3). The specific activity of the enzyme was 430 IU/mg.
Conclusion: The results of the present work form the basis for a new engineered form of ErA for future therapeutic use, which could be extended with crystallographic studies.
Keywords
Characterization
cloning
ErA
Erwinia carotovora
Escherichia coli BL21 (DE3)
L-asparaginase
purification
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Advanced Biomedical Research
Volume 2014, February
February 2014
Pages 1-6