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

## TL;DR

This paper describes the production and recovery of a new dual-function enzyme from bacteria, which could be used for amino acid depletion therapy.

## Contribution

A scalable workflow for producing and refolding a bifunctional chimeric enzyme from nonclassical inclusion bodies is demonstrated.

## Key findings

- The chimeric enzyme 63N-hC_hARG1 was expressed in E. coli with a preparative yield of 25.8 mg mL–1.
- Refolding restored 0.22 U mL–1 of l-asparaginase and 4.66 U L–1 of arginase 1 activity.
- In-silico modeling supported the structural compatibility and dual functionality of the chimeric enzyme.

## Abstract

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.