Designing strategies for high-level production of peptide-N-glycosidase F (PNGase F) from Flavobacterium meningosepticum using high cell density fed-batch culture of E. coli.

Abstract

Protein N-glycosylation is a crucial post-translational modification that regulates cellular processes such as cell signalling, development, and autophagy. Abnormalities in protein glycosylation can manifest in life-threatening conditions, such as cancer. N-glycan analysis is crucial for determining the underlying cause of disease. The characterization of N-linked glycans is achieved through the removal of the carbohydrate moiety using deglycosylating enzymes. Peptide-N-glycosidase F (PNGase F) is a glycoamidase that hydrolyzes the amide bond in glycosamide by specifically cleaving at the innermost N-acetylglucosamine (GlcNAc). Although PNGase F has significant therapeutic applications, its widespread commercial use is limited by its high cost. This study focused on heterologous expression of Flavobacterium meningosepticum PNGase F in E. coli BL21 (DE3) and SHuffle^® cells, in which the protein was partially soluble. The E. coli SHuffle^® cells yielded 210.41 mg/L of PNGase F in TB glycerol medium in shake flasks, with a corresponding Y_P/X of 47.20 mg/g DCW. Expression studies in E. coli BL21 (DE3) cells yielded inclusion bodies (IBs) that were solubilized, yielding activity comparable to that of the soluble form. Furthermore, the fed-batch cultivation in E. coli BL21 (DE3) cells produced 5.87 g/L of IBs with a final OD₆₀₀ of 176. Therefore, this study investigates the potential of alternative E. coli hosts as cost-effective production platforms for PNGase F.