Overcoming challenges in the production of a human-like L-asparaginase: advanced strategies for expression and purification.

Human l-asparaginase 1 (hASNase1) is a promising next-generation candidate for Acute Lymphoblastic Leukemia (ALL) therapy due to its reduced immunogenicity and superior physiological compatibility. However, its recombinant production remains challenging, as expression in Escherichia coli typically results in insoluble aggregates and strong association with host chaperones, limiting biochemical characterization. Here, we established an optimized workflow combining mild induction conditions with tailored solubilization and purification strategies. Incubation with l-asparagine followed by ATP-mediated washing during affinity chromatography significantly improved protein recovery while reducing chaperone co-purification. This approach enabled the isolation of catalytically active hASNase1 displaying allosteric regulation and cooperative substrate binding, consistent with its proposed tetrameric organization. Complementary in silico analyses identified aggregation-prone regions, potentially providing structural insight into the observed expression challenges. Collectively, this study contributes to the development of improved recombinant production strategies for challenging human enzymes in prokaryotic systems.