Expression, Solubilization, and Refolding Recovery of a Novel l -Asparaginase–Arginase 1 Chimera from E. coli Inclusion Bodies

The preparative expression, purification, and refolding of a novel bifunctional chimeric enzyme, 63N-hC_hARG1, engineered for dual amino acid depletion therapy, are described. A guinea pig–human l-asparaginase hybrid (63N-hC) was fused to human arginase 1 through a rigid helical linker, and the codon-optimized gene was expressed in Escherichia coli BL21(DE3). Nonclassical inclusion bodies (IBs) were obtained, exhibiting activities of 2.16 ± 0.04 U mL–1 for 63N-hC and 13.42 ± 0.09 U L–1 for hARG1, with a preparative yield of 25.8 ± 0.6 mg mL–1 from the lysate. After solubilization in 8 M urea, size-exclusion chromatography and reverse-dilution refolding were performed, restoring activities to 0.22 ± 0.05 U mL–1 and 4.66 ± 0.9 U L–1, respectively. Structural compatibility and potential dual functionality were supported by in-silico modeling and molecular docking. A scalable workflow for expression optimization and functional recovery of multimeric chimeric proteins from IBs is thus demonstrated, highlighting key parameters governing the refolding of complex fusion proteins and the value of nonclassical IBs as reservoirs for therapeutic enzyme production.