Investigation into the efficiency of diverse N-linking oligosaccharyltransferases for glycoengineering using a standardised cell-free assay.
Microb Biotechnol
; 17(6): e14480, 2024 Jun.
Article
em En
| MEDLINE
| ID: mdl-38858807
ABSTRACT
The application of bacterial oligosaccharyltransferases (OSTs) such as the Campylobacter jejuni PglB for glycoengineering has attracted considerable interest in glycoengineering and glycoconjugate vaccine development. However, PglB has limited specificity for glycans that can be transferred to candidate proteins, which along with other factors is dependent on the reducing end sugar of glycans. In this study, we developed a cell-free glycosylation assay that offers the speed and simplicity of a 'yes' or 'no' determination. Using the assay, we tested the activity of eleven PglBs from Campylobacter species and more distantly related bacteria. The following assorted glycans with diverse reducing end sugars were tested for transfer, including Streptococcus pneumoniae capsule serotype 4, Salmonella enterica serovar Typhimurium O antigen (B1), Francisella tularensis O antigen, Escherichia coli O9 antigen and Campylobacter jejuni heptasaccharide. Interestingly, while PglBs from the same genus showed high activity, whereas divergent PglBs differed in their transfer of glycans to an acceptor protein. Notably for glycoengineering purposes, Campylobacter hepaticus and Campylobacter subantarcticus PglBs showed high glycosylation efficiency, with C. hepaticus PglB potentially being useful for glycoconjugate vaccine production. This study demonstrates the versatility of the cell-free assay in rapidly assessing an OST to couple glycan/carrier protein combinations and lays the foundation for future screening of PglBs by linking amino acid similarity to glycosyltransferase activity.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Hexosiltransferases
/
Proteínas de Membrana
Idioma:
En
Revista:
Microb Biotechnol
Ano de publicação:
2024
Tipo de documento:
Article
País de afiliação:
Reino Unido