# Investigation of the Impact of a Protein Source on the Purification of l‑Asparaginase Type II from Escherichia coli

## TL;DR

This study compares two methods for purifying l-asparaginase from E. coli, finding that broth purification yields higher amounts with less contamination.

## Contribution

The study introduces a comparative analysis of two purification methods for l-asparaginase, highlighting differences in yield and contamination levels.

## Key findings

- Broth purification yielded 66.3 mg/L of l-asparaginase with higher specific activity compared to cell pellet lysate.
- Proteomic analysis showed fewer host cell proteins in the broth-purified enzyme.
- Broth purification demonstrated better reproducibility and lower contamination than cell pellet lysate.

## Abstract

Escherichia coli
l-asparaginase
type II (EcA2) is essential for treating childhood acute lymphoblastic
leukemia (ALL), improving survival rates since its introduction. After
the expiration of its original patents, interest in producing biosimilars
has increased, particularly to reduce treatment-related side effects.
In this study, we compared two production methods for EcA2, purifying
the enzyme from broth and from the soluble fraction of the cell pellet
lysate. Using a converging purification workflow, we obtained 66.3
(±2.3) mg/L of l-asparaginase from broth and 29.2 (±4.6)
mg/L from the cell pellet lysate, with specific activities of 136.3
(±13.3) and 123.5 (±10.3) IU/mg, respectively. Both versions
showed similar three-dimensional structures, thermal stability, and
specific activity, with no significant differences in performance.
Proteomic analysis revealed that purification from broth resulted
in fewer host cell proteins than purification from the cell pellet
lysate. Our results further suggest that the purification process
from cell lysate is more susceptible to variability than purification
from the broth. These findings demonstrate that while both production
methods yield comparable enzymes in terms of structure and activity,
purifying from broth may offer advantages in terms of lower contamination
and better reproducibility.