20-09-2018 дата публикации
Номер: US20180265910A1
Provided herein is an α-fucosidase that can cleave a conjugate comprising an N-glycan and a label where the label is added by amine reactive chemistry. The α-fucosidase also has an accelerated reaction time using Schiff base labeled N-glycans compared with BKF. A reaction mix, enzyme mix and kit comprising the α-fucosidase are provided, as well as a method for analyzing glycoproteins. The α-fucosidase finds particular use in analyzing the N-glycans of therapeutic glycoproteins. 1. A reaction mix comprising:(a) an α-L-fucosidase having an amino acid sequence that is at least 90% identical to amino acids 23-359 of sequence of SEQ ID NO:1 ; and(b) a conjugate comprising an N-glycan and a label.2. The reaction mix of claim 1 , wherein the label comprises a fluorophore and/or a charge tag.3. The reaction mix of claim 1 , wherein the N-glycan of the conjugate comprises a core fucose.4. The reaction mix of claim 1 , wherein the N-glycan is linked to the label via the reducing end of the N-glycan.5. The reaction mix of claim 1 , wherein the N-glycan is linked to the label via amine reactive chemistry or by a Schiff base condensation reaction.6. The reaction mix of claim 1 , wherein the N-glycan is from a therapeutic glycoprotein.7. The reaction mix of claim 1 , wherein the reaction mix further comprises: α2-3 neuraminidase S,', 'α2-3,6,8,9 neuraminidase A,', 'α1-3,4,6 galactosidase,', 'β1-4 galactosidase,', 'β-N-acetylglucosaminidase S, and', 'α1-2,3,6 mannosidase., '(c) one or more exoglycosidases selected from the group consisting of8. The reaction mix of claim 1 , wherein the reaction mix has a pH in the range of pH 3.0 to pH 5.5.9. An enzyme mix comprising:(a) an α-fucosidase having an amino acid sequence that is at least 90% identical to amino acids 23-359 of sequence of SEQ ID NO:1; and α2-3 neuraminidase S,', 'α2-3,6,8,9 neuraminidase A,', 'α1-3,4,6 galactosidase,', 'β1-4 galactosidase,', 'β-N-acetylglucosaminidase S, and', 'α1-2,3,6 mannosidase., '(b) one or more ...
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