Glycan-Specific Analytical Tools
AssigneesUniversity of Georgia Research Foundation, Inc., Glycosensors and Diagnostics, LLC
InventorsRobert J. Woods, Loretta Yang
Life of this patent
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Abstract
Provided are lectenz molecules, which are mutated carbohydrate processing enzyme enzymes that are catalytically inactive and that have had their substrate affinity increased by at least 1.2 fold. Further provided are methods for making and methods of using such lectenz. Additional mutated proteins following the lectenz approach are further provided.
Claims43
A lectenz comprising a carbohydrate processing enzyme, said enzyme having one or more mutations that:
- eliminate catalytic activity of the enzyme;
- and
- (ii) increase the affinity of the inactivated enzyme to its substrate as compared to a wild-type enzyme.
A method for generating a lectenz, wherein the lectenz comprises a carbohydrate processing enzyme, said enzyme having one or more mutations that:
- (i) eliminate catalytic activity of the enzyme;
- and
- (ii) increase the affinity of the enzyme to its substrate, as compared to a wild-type enzyme;
- the method comprising:
- (a) analyzing a sequence of a carbohydrate-processing enzyme for one or more amino acid residues that, when mutated, could inactivate the enzyme;
- (b) performing a computational simulation to predict binding energies of the glycosidase-glycan complex, wherein the glycosidase has at least one mutated amino acid identified in step (a);
- (c) subdividing the residues on the basis of their predicted interaction energies into two groups, a first group of residues that are essential to defining the specificity of the enzyme, and a second group of residues that are proximal to the substrate but not essential to defining specificity;
- (d) testing glycosidases comprising mutations identified in steps (a) and (b) and (c) for their ability to form the glycosidase-glycan complex;
- and
- (e) identifying mutants from step (d) that exhibit binding affinities to the glycan that are at least 1.2-fold greater than those of WT glycosidase.
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A method for generating a novel biomolecule, wherein the biomolecule comprises a carbohydrate-binding protein, said protein having one or more mutations that:
- (i) Increase the affinity of the protein to its substrate, as compared to a wild-type protein;
- (ii) Have substantially no alteration in the specificity of the protein;
- the method comprising:
- (a) Performing a computational simulation to predict binding energies of the carbohydrate-protein complex, wherein the protein;
- (b) Subdividing the residues on the basis of their predicted interaction energies into two groups, a first group of residues that are essential to defining the specificity of the protein, and a second group of residues that are proximal to the carbohydrate ligand but not essential to defining specificity;
- (c) Testing proteins comprising mutations identified in steps (a) and (b) for their ability to form the protein-glycan complex;
- and
- (d) Identifying mutants from step (c) that exhibit binding affinities to the glycan that are at least 1.2-fold greater than those of WT protein.
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Dependencies are inferred from the claim text.