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<p>Protein <i>N</i>-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. <i>N</i>-glycan analysis is crucial for determining the underlying cause of disease. The characterization of <i>N</i>-linked glycans is achieved through the removal of the carbohydrate moiety using deglycosylating enzymes. Peptide-<i>N</i>-glycosidase F (PNGase F) is a glycoamidase that hydrolyzes the amide bond in glycosamide by specifically cleaving at the innermost <i>N</i>-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 <i>Flavobacterium meningosepticum</i> PNGase F in <i>E. coli</i> BL21 (DE3) and SHuffle<sup>®</sup> cells, in which the protein was partially soluble. The <i>E. coli</i> SHuffle<sup>®</sup> cells yielded 210.41&#xa0;mg/L of PNGase F in TB glycerol medium in shake flasks, with a corresponding Y<sub>P/X</sub> of 47.20&#xa0;mg/g DCW. Expression studies in <i>E. coli</i> 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 <i>E. coli</i> BL21 (DE3) cells produced 5.87&#xa0;g/L of IBs with a final OD<sub>600</sub> of 176. Therefore, this study investigates the potential of alternative <i>E. coli</i> hosts as cost-effective production platforms for PNGase F.</p> Graphical abstract <p></p>

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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

  • 期刊论文

  • 发表日期：

    2026年5月2日

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  • Shilpa Mohanty,
  • Babbal,
  • Shivani Chauhan,
  • Mohini Talwar,
  • Shubham Sharma,
  • Priya Maravi,
  • Yogender Pal Khasa

摘要

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 YP/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.

Graphical abstract