Performance Testing on Engineered Glycoproteins using FucosEXO®

Performance Activity

Rapid and complete defucosylation of O-glycans on TNFR proteins, outperforming alternative fucosidases across glycoengineered substrates.

Fucosylation of O-glycans is involved in the synthesis of functionally important glycan epitopes such as blood group antigens and the Lewis structures. Analysis of glycoproteins modified with such complex glycan structures can be challenging and requires efficient and specific enzymatic tools.

TNFR proteins carry up to 11 O-glycan structures, and we engineered these substrates to decorate them with different linkages of fucose. We then tested FucosEXO on these glycoengineered TNFR proteins and compared the activity to other commercially available fucosidases. FucosEXO is able to defucosylate all three TNFR proteins within 1 hour, while treatment with the other fucosidases only led to partial removal of fucose or no removal at all.

Improved removal of a1-2, a1-3 and a1-4 linked fucose compared to other available fucosidases

Performance testing of FucosEXO against other commercially available fucosidases. TNFR proteins were pretreated with PNGase F to remove N-glycans before being glycoengineered to add different linkages of fucose to the O-glycans. These glycoengineered TNFR proteins were incubated with FucosEXO for 1h at 37°C or with other fucosidases according to the manufacturers recommendations. The resulting proteins were analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm).

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