Upstream endotoxin removal in recombinant protein purification: an optimised pre-cell disruption strategy for reducing endotoxin contamination of inclusion bodies in Escherichia coli.

Endotoxin contamination remains a critical challenge in recombinant protein production using Gram-negative bacteria, particularly for intracellularly expressed, positively charged proteins prone to lipopolysaccharide (LPS) binding. This study introduces an efficient upstream strategy for LPS removal before the cell disruption, aimed at reducing the endotoxin contamination load in the downstream purification process. A recombinant cationic protein (named as 4mer-S3-DP) was expressed in Escherichia coli (E. coli) BL21 (DE3), and biomass was treated with varying concentrations and incubation times of Tris-HCl and EDTA, as defined by a central composite design (CCD) of response surface methodology (RSM). Endotoxin levels in inclusion bodies (IBs) were quantified using a chromogenic Limulus Amebocyte Lysate (LAL) assay, and statistical modelling was performed to optimize treatment conditions. The final quadratic model demonstrated high predictive accuracy, and response surface analysis revealed that both insufficient and excessive treatment conditions could increase endotoxin contamination. Optimal conditions, consisting of 70 mM Tris-HCl for 30 min and 60 mM EDTA for 5 min, achieved a 2.7-log reduction in endotoxin levels (99.8%) without compromising protein integrity, as confirmed by SDS-PAGE analysis. Experimental validation of the optimum criteria predicted by this model showed no significant difference between predicted and observed endotoxin levels of IBs. This upstream intervention as a simple and effective endotoxin removal approach before the cell lysis could be adaptable to other recombinant proteins expressed in Gram-negative bacterial.