Design and Activity Assay of β1,3-Galactosyltransferase (CgtB) Mutants from Bacterium Campylobacter jejuni

Abstract:

β1,3-galactosyltransferase(CgtB) from bacterium Campylobacter jejuni is mainly responsible for the second elongation of proteins O-glycosylation by transferring a galactose to O-linked N-acetylgalacosamine (O-GalNAc). Presently, CgtB has been widely applied in the synthesis of O-glycoproteins. To develop a recognition tool for O-GalNAc modification, the CgtB protein was used for mutant design. Cgt1 was generated by deleting 30 amino acids from both the N- and C-terminal regions at the gene level. Cgt1 was constructed in pMal-c5x expression plasmid and expressed in the Escherichia coli. The soluble Cgt1 was purified by affinity chromatography with maltose binding protein(MBP) column and the activity assay was carried out by glycan binding activity. The results suggested that the soluble recombinant Cgt1 with the molecular weight of approximately 70 000 could be successfully expressed in the system of Escherichia coli. The recombinant Cgt1 possessed the activity of binding O-GalNAc modified proteins. For different standard glycoproteins, Cgt1 only recognized mucin containing O-GalNAc modification. For complex cellular samples, Cgt1 could specifically recognize intracellular O-GalNAc modification. The developed recognition tool for O-GalNAc modification in this research will provide a foundation for further research on the function of O-GalNAc modification.