Site-Specific Modification of Recombinant Proteins: A Novel Platform for Modifying Glycoproteins Expressed in E. coli

Detailed analysis of the glycosylated target mucin peptide with eight potential glycosylation sites has not been shown.

“However, the detailed analysis of the glycosylated target mucin peptide with eight potential glycosylation sites has not been shown [9].”

In a previously published expression system, an N-terminally truncated GalNAc T2 under the T7 promoter was expressed with the 4-epimerase from P. aeruginosa including its endogenous promoter in E. coli SHuffle®T7 Express without IPTG induction.

“In the expression system published previously, an N-terminally truncated version of GalNAc T2, inserted under the control of the T7 promoter, has been expressed in combination with the 4-epimerase derived from P. aeruginosa including its endogenous promoter in E. coli SHuffle®T7 Express without IPTG induction [9].”

In vivo O-glycosylation of a glutathione S-transferase-mucin peptide fusion in an E. coli SHuffle® T7 express strain co-expressing an N-terminal truncated version of GalNAc-T2, in combination with the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa, has been indicated based on Western blot analysis using horseradish peroxidase-conjugated Vicia villosa lectin.

“The in vivo O-glycosylation of a glutathione S-transferase-mucin peptide fusion in an E. coli SHuffle® T7 express strain co-expressing an N-terminal truncated version of GalNAc-T2, in combination with the UDP-GlcNAc 4-epimerase WbpP from Pseudomonas aeruginosa has been indicated based on Western blot analysis using horseradish peroxidase-conjugated Vicia villosa lectin [9].”

O-glycosylation of eukaryotic target sites using a truncated form of human GalNAc-T2 has been shown without altering the target peptide sequence.

“In contrast, O-glycosylation of eukaryotic target sites using a truncated form of the human glycosyltransferase GalNAc-T2 has been shown without altering the target peptide sequence [6, 9, 14, 15].”

The absence of necessary activated sugar substrate UDP-GalNAc in laboratory E. coli strains requires co-expression of an additional UDP-GlcNAc 4-epimerase to generate UDP-GalNAc from UDP-GlcNAc.

“The absence of the necessary activated sugar substrate (UDP-GalNAc) in laboratory E. coli strains requires the co-expression of an additional UDP-GlcNAc 4-epimerase to generate UDP-GalNAc from UDP-GlcNAc [9, 20].”

In vivo glycosylation has been shown.

“In vivo glycosylation has also been shown [9].”

LC/MS analysis of an anti-TNF-α antibody fragment fusion to the 15 amino acid mucin-derived small peptide CDNKPAPGSTAPPAA revealed an increase in molecular weight of 203 Da, consistent with a single GalNAc moiety.

“The analysis of an anti-TNF-α antibody fragment fusion to the 15 amino acid mucin-derived small peptide CDNKPAPGSTAPPAA by LC/MS revealed an increase in molecular weight of 203 Da, consistent with the presence of a single GalNAc moiety [9].”