Optimized E. coli fed-batch culture yields **210.41 mg/L** soluble **PNGase F** for N-glycan analysis

Protein N-glycosylation is a fundamental post-translational modification governing critical cellular processes like cell signaling, development, and autophagy. Dysregulation of protein glycosylation is implicated in severe conditions, including various forms of cancer. Accurate N-glycan analysis is therefore indispensable for understanding disease mechanisms, developing diagnostics, and ensuring the quality of biopharmaceutical products. Peptide-N-glycosidase F (PNGase F) is an enzyme widely used to cleave N-glycans from glycoproteins, making it a cornerstone tool in glycobiology research and industrial applications. Efficient, high-yield production of active PNGase F is essential to support these diverse needs.

Researchers designed strategies for high-level production of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum using high cell density fed-batch culture in E. coli. Initial expression studies utilized E. coli SHuffle® cells grown in TB glycerol medium in shake flasks. Further investigations included expression in E. coli BL21 (DE3) cells. The primary endpoints were the total yield of PNGase F (mg/L) and specific yield (mg/g DCW), with activity measurements performed on both soluble protein and solubilized inclusion bodies.

Initial expression in E. coli SHuffle® cells cultivated in TB glycerol medium within shake flasks achieved a substantial yield of 210.41 mg/L of PNGase F. This corresponded to a specific yield (YP/X) of 47.20 mg/g DCW. Subsequent expression studies using E. coli BL21 (DE3) cells resulted in the formation of inclusion bodies (IBs).