Acute lymphoblastic leukaemia (ALL) affects lymphoblastic cells and is the most common neoplasm during childhood. Among the pharmaceuticals used in the treatment protocols for ALL, Asparaginase (ASNase) fromEscherichia coli(EcAII) is an essential biodrug. Meanwhile, the use of EcAII in neoplastic treatments causes several side effects, such as immunological reactions, hepatotoxicity, neurotoxicity, depression, and coagulation abnormalities. Commercial EcAII is expressed as a recombinant protein, similar to novel enzymes from different organisms; in fact, EcAII is a tetrameric enzyme with high molecular weight (140 kDa), and its overexpression in recombinant systems often results in bacterial cell death or the production of aggregated or inactive EcAII protein, which is related to the formation of inclusion bodies. On the other hand, several commercial expression strains have been developed to overcome these expression issues, but no studies on a systematic evaluation of theE. colistrains aiming to express recombinant asparaginases have been performed to date. In this study, we evaluated eleven expression strains at a low temperature (16 degrees C) with different characteristics to determine which is the most appropriate for asparaginase expression; recombinant wild-type EcAII (rEcAII) was used as a prototype enzyme and the secondary structure content, oligomeric state, aggregation and specific activity of the enzymes were assessed. Structural analysis suggested that a correctly folded tetrameric rEcAII was obtained using ArcticExpress (DE3), a strain that co-express chaperonins, while all other strains produced poorly folded proteins. Additionally, the enzymatic assays showed high specific activity of proteins expressed by ArcticExpress (DE3) when compared to the other strains used in this work.