Abstract
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) from Escherichia 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 the E. coli strains aiming to express recombinant asparaginases have been performed to date. In this study, we evaluated eleven expression strains at a low temperature (16 °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.
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Acknowledgements
This work was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant Numbers: 2011/13500-6, 2013/08617-7, 2014/22039-9, 2016/19245-1, 2017/19942‐7, 2017/20291‐0, 2017/25272-4 and 2019/04054-4).
Supporting information
Supplementary Fig. 1—SDS-PAGE containing the rEcAII purification expressed in BL21 (DE3), Tuner (DE3) and C43 (DE3) strains at 37ºC.
Supplementary Fig. 2—SDS-PAGE containing the rEcAII purification expressed in different strains of Escherichia coli at 16ºC.
Supplementary Fig. 3—Normalized rEcAII concentrations after IMAC purification shown by SDS-PAGE.
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de Moura, W.A.F., Schultz, L., Breyer, C.A. et al. Functional and structural evaluation of the antileukaemic enzyme l-asparaginase II expressed at low temperature by different Escherichia coli strains. Biotechnol Lett 42, 2333–2344 (2020). https://doi.org/10.1007/s10529-020-02955-5
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DOI: https://doi.org/10.1007/s10529-020-02955-5