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US 9,926,612 B2Granted
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 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.
Claims18
A method for generating a lectenz comprising an inactivated mutated carbohydrate-processing enzyme having enhanced affinity for its substrate compared to a corresponding wild-type carbohydrate-processing enzyme, the method comprising:
- (a) providing a 3D structure of an enzyme-substrate complex comprising a substrate bound to a catalytically inactive mutant carbohydrate-processing enzyme or a corresponding wild-type carbohydrate-processing enzyme, wherein the catalytically inactive carbohydrate-processing enzyme comprises an amino acid sequence comprising at least one inactivating mutation that eliminates catalytic activity of the enzyme;
- (b) performing a computational simulation on the enzyme-substrate complex to predict the per-residue contributions to total interaction energy (ΔEMM) and/or total binding free energy (ΔGBinding) for amino acid residues of the enzyme;
- (c) analyzing the per-residue energetic contributions to identify at least one amino acid residue as a potential mutation site for enhancing binding affinity of the inactivated enzyme for its substrate as compared to a wild-type enzyme, wherein an amino acid residue is identified as a potential mutation site for enhancing binding activity when
- (i) for an amino acid residue located within 5 Å of the substrate in the enzyme-substrate complex, the per-residue contribution of the amino acid residue to at least one of ΔEMM or ΔGBinding is ≥−0.7 kcal/mol;
- and
- (ii) for an amino acid residue located more than 5 Å from the substrate in the enzyme-substrate complex, the per-residue contribution of the amino acid residue to at least one of ΔEMM or ΔGBinding is ≥0.0 kcal/mol;
- (d) expressing a library of inactivated mutated carbohydrate-processing enzymes, each inactivated mutated enzyme comprising a plurality of amino acid mutations, wherein
- each inactivated mutated enzyme comprises at least one inactivating mutation that eliminates catalytic activity of the enzyme;
- and
- each inactivated mutated enzyme further independently comprises at least one potential affinity-enhancing mutation at a site identified in step (c);
- (e) assaying the inactivated mutated enzymes of step (d) for their ability to form the enzyme-substrate complex;
- and
- (f) identifying inactivated mutated enzymes from step (e) that exhibit binding affinities to the substrate that are at least 1.2-fold greater than those of the wild-type carbohydrate-processing enzyme.
10 more elements
A method for generating a novel biomolecule, wherein the biomolecule comprises a carbohydrate-binding protein having one or more mutations that:
- (i) increase the affinity of the protein to its carbohydrate ligand, as compared to a wild-type protein;
- and
- (ii) have substantially no alteration in the specificity of the protein;
- the method comprising:
- (a) performing a computational simulation on the carbohydrate-protein complex to predict per-residue contributions to total interaction energy (ΔEMM) or total binding free energy (ΔGBinding) for amino acid residues of the protein;
- (b) analyzing the per-residue energetic contributions to identify at least one amino acid residue as a potential mutation site for enhancing binding affinity of the protein for the carbohydrate ligand as compared to a wild-type protein, wherein an amino acid residue is identified as a potential mutation site for enhancing binding affinity when the amino acid residue is located within 5 Å of the carbohydrate ligand in the carbohydrate-protein complex, and the per-residue contribution of the amino acid residue to at least one of ΔEMM or ΔGBinding is ≥−0.7 kcal/mol;
- (c) expressing a library of mutant carbohydrate-binding proteins, wherein each mutant carbohydrate-binding protein independently comprises at least one potential affinity-enhancing amino acid substitution that replaces an amino acid residue identified in step (b);
- (d) assaying the mutant proteins of step (c) for their ability to form the carbohydrate-protein complex and for binding specificity;
- and
- (e) identifying mutant proteins from step (d) that exhibit binding affinities to the carbohydrate that are at least 1.2-fold greater than those of wild-type protein.
7 more elements
Dependencies are inferred from the claim text.