Comprehensive Glycosylase Genetic Analysis Service

CD BioGlyco is constantly enriching and improving our Glycan Display Platform. Glycophage Display is used for the analysis of Glycosylases, Glycoarray, Antibody Development, Epitope Mapping, and Biomarker Development. In order to provide comprehensive support for our clients' glycobiology research, we have developed a glycosylation enzyme genetic analysis service based on glycophage display. Analysis of glycosylases and their genetic variants by glycophage display provides an opportunity to make glycosylation a tunable mechanism. Glycosylation enzymes that can be analyzed include glycosyltransferases, flippases, etc., involved in N-linked glycosylation, O-linked glycosylation, and others.

We insert genes encoding glycoproteins or genes encoding glycosylation components into the phage genome so that the relevant glycoproteins bind to phage shell proteins (g3p) to form fusion proteins. Escherichia coli carrying phages encoding fusion proteins with plasmids encoding different glycosylation enzyme variants are subsequently cultured. A specific phage library displaying different glycoproteins is finally constructed by this method. We use this library to analyze the genetic variation of glycosylation enzymes and so on. The phage particles obtained by this method also provide a physical link between glycosylated receptor proteins and glycosylase genes.

The experimental procedure for glycophage display-based glycosylase genetic analysis consists of the following steps:

• Construction of phage libraries
  We construct diverse phage libraries by fusing genes encoding glycoproteins or genes encoding glycosylation components with genes encoding phage shell proteins (g3p) using NKK parsimony primers. After construction, we screen and validate all phages constructed by DNA sequencing.
• Expression of fusion protein and purification of phage
  We culture phages expressing the fusion protein using E. coli carrying a plasmid encoding a glycosylase as a host system. Phage purification is subsequently performed using polyethylene glycol/NaCl.
• Biopanning of phages
  Non-glycosylated phage may be present in the obtained phage, which needs to be biopanning. We use lectin affinity chromatography to distinguish between non-glycosylated and glycosylated phage. The screened glycosylated phages are infected with E. coli and confirmed by DNA sequencing.
• Western blot and functional analysis
  We analyze fusion proteins by western blot. We also use bioassays and structural analysis to reveal the molecular determinants of glycosylase function. Directed evolutionary studies of glycosylases are also supported by the construction of different gene variants.

Fig.1 Process of glycophage display-based glycosylase genetic analysis. (CD BioGlyco)

Publication Data

Applications of Glycophage Display-based Glycosylase Genetic Analysis

• Glycophage display-based glycosylase genetic analysis provides a comprehensive approach to studying genetic variation in glycosylase, enabling researchers to explore the impact of genetic variation on enzyme function, substrate specificity
• Glycophage display-based glycosylase genetic analysis is used to identify and analyze glycosylase variants with altered enzyme activity.
• Glycophage display-based glycosylase genetic analysis is used to analyze the association between glycosylase and disease.

Highlights of Us

• We meet the needs of our clients regarding glycosylation enzyme research by constructing diverse phage libraries.
• We improve the recovery rate of the lectin capture method in phage screening by various methods.
• The specific experimental procedures of our glycophage display-based glycosylase genetic analysis will be changed and adjusted according to the requirements of our clients and the results of actual experiments.

CD BioGlyco constructs glycophage display technology for antibody development, glycosylation enzyme gene analysis, etc., covering a wide range of library types. We provide flexible construction, screening, and analysis services according to the needs of our clients' applications. If you are interested in our glycophage display-based glycosylase genetic analysis service, please feel free to contact us to tell us the purpose of your experiment.