Glycosyltransferases (GTs)-based Cell Surface Chemoenzymatic Glycoengineering Service

Glycosyltransferases (GTs)-based Cell Surface Chemoenzymatic Glycoengineering Service at CD BioGlyco

The chemoenzymatic glycoengineering (CGE) method is highly specific and controls the attachment of acceptor, donor sugars, and sugar moieties. It utilizes GTs, to catalyze the transfer of sugar residues from their activated sugar nucleotide donors to the acceptor. CD BioGlyco has professional Technologies for Cell Surface Glycoengineering, and we provide professional GTs-based cell surface chemoenzymatic glycoengineering services.

• Enzyme expression and purification services

β1-4-galactosyltransferase named NmLgtB from Neisseria meningitidis catalyzes the transfer of Gal residues from the sugar nucleotide UDP-Gal to the receptor. CD BioGlyco provides various enzyme expression and purification services, which mainly include the following steps.

• Gene cloning: We clone the enzyme genes (NmLgtB) into a suitable expression vector (for example plasmid).
• Expression host construction: We transform the recombinant plasmid into an expression host strain, such as Escherichia coli.
• Preculture and induced expression: We preculture the expression host strain in an appropriate medium, and then add appropriate inducers to induce the expression of the target enzyme.
• Cell harvest: We culture cells to the appropriate growth phase, then harvest cells. We use appropriate methods (such as sonication, or chemical lysis agents) to disrupt the cells and obtain a cell lysate.
• Purification: We use affinity chromatography and select an appropriate method for purification based on the characteristics of the enzyme.
• Cell surface chemoenzymatic glycoengineering services

CD BioGlyco incubates the purified enzyme with cells at 37°C to perform cell surface chemoenzymatic glycoengineering services. NmLgtB is incubated in the presence of UDP-6N₃Gal. After treating the cells with enzyme, we wash and fix the cells, then perform click chemistry reactions with biotin alkyne. Thus, azide-modified Gal sugars are incorporated into cell surface glycans.

• Modification analysis services

At the same time, CD BioGlyco provides professional analysis and verification for modified cells. We use a variety of technical methods such as mass spectrometry, flow cytometry, and cell immunostaining to confirm whether the required glycosyl structure has been successfully introduced into the cell surface.

Fig.1 GTs-based cell surface chemoenzymatic glycoengineering services. (CD BioGlyco)

Publication

Technology: Chemoenzymatic glycoengineering approach

Journal: Biology Open

IF: 2.643

Published: 2017

Results: Through chemoenzymatic glycoengineering technology, the authors successfully used NmLgtB to effectively integrate Gal residues into Lec8 cells. Under cell culture-like conditions, GTs incorporated synthetic sugars into the cell surface within 20 minutes. The study showed that GTs previously characterized in a one-pot multienzyme reaction can also glycosylate living cells.

Fig.2 Cell labeling results of GTs. (Almaraz & Li, 2017)

Applications

• Protein modification: Introduce specific GTs to the cell surface to achieve glycosyl modification of the target protein.
• Antibody modification: Using cell surface chemoenzymatic glycoengineering technology, different types of sugar groups, such as mannose, fucose, etc., are introduced into the Fc region of the antibody, thereby changing its immune activity, stability, and pharmacological properties.
• Biosensor: Using cell surface chemoenzymatic glycoengineering technology, specific GTs are introduced to the cell surface and combined with appropriate probes, which construct biosensors with high sensitivity and selectivity for detecting the biological activity of target molecules.

Advantages

• CD BioGlyco optimizes the conditions of GTs catalyzed reactions, such as pH value, temperature, etc., to improve the catalytic efficiency and selectivity of the enzyme, thereby achieving efficient GTs-based cell surface chemoenzymatic glycoengineering services.
• CD BioGlyco has a wide range of glycosyltransferase resources and outstanding technology platforms to meet the needs of different substrate modifications based on the cell surface.
• CD BioGlyco conducts cutting-edge glycoengineering research, discovers and develops new enzymes and modification methods, and provides clients with innovative solutions.

CD BioGlyco has extensive expertise and experience in glycosyltransferase engineering and glycochemistry and provides excellent GTs-based cell surface chemoenzymatic glycoengineering services. Please feel free to contact us if you would like to inquire about specific service details.