Covalent tethering of functional groups to proteins
AssigneePromega Corporation
InventorsKeith V. Wood, Dieter Klaubert, Georgyi V. Los, Robert F. Bulleit, Mark McDougall, Chad Zimprich
Life of this patent
Estimating the term…
Abstract
A mutant hydrolase optionally fused to a protein of interest is provided. The mutant hydrolase is capable of forming a bond with a substrate for the corresponding nonmutant (wild-type) hydrolase which is more stable than the bond formed between the wild-type hydrolase and the substrate. Substrates for hydrolases comprising one or more functional groups are also provided, as well as methods of using the mutant hydrolase and the substrates of the invention. Also provided is a fusion protein capable of forming a stable bond with a substrate and cells which express the fusion protein.
Claims40
A method for preparing a compound of the formula biotin-Linker-A—X comprising coupling a compound of formula biotin-Y with a compound of formula Z-Linker-A—X, wherein Y and Z are groups that can react to link biotin to Linker-A—X, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates biotin and X by at least 11 atoms, wherein A—X is a substrate for a dehalogenase, wherein A is (CH2)n and n=2-10, wherein X is a halogen, wherein biotin is a functional group is capable of being coupled through its carboxy terminus to the linker, and wherein biotin-Y is an activated ester of biotin and wherein Z is an amine suitable to react with the activated ester to form an amide bond.
A method for preparing a compound of the formula biotin-Linker-A—X wherein the Linker comprises an amide bond comprising coupling a corresponding activated ester with a corresponding amine to provide the compound of formula biotin Linker-A—X, wherein biotin is a functional group, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein A—X is a substrate for a dehalogenase, wherein A is (CH2)n and n=2-10, and wherein X is a halogen.
A compound of formula (I):
- biotin-linker-A—X, wherein biotin is a functional group, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates biotin and X by at least 11 atoms, wherein A is (CH2)n and n=4-10, wherein A—X is a substrate for a dehalogenase, and wherein X is a halogen, wherein the biotin functional group is coupled through its carboxy terminus to the linker.
A compound of formula (I):
- biotin-linker-A—X, wherein biotin is a functional group, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates biotin and X by at least 11 atoms, wherein A is (CH2)n and n=2-10, wherein A—X is a substrate for a dehalogenase, wherein X is a halogen, and wherein the biotin functional group is coupled through its carboxy terminus to the linker.
A compound of formula (II):
A method to detect or determine the presence or amount of a mutant hydrolase, comprising:
- a) contacting a mutant hydrolase with a hydrolase substrate which comprises one or more biotin functional groups, wherein the mutant hydrolase comprises at least one amino acid substitution relative to a corresponding wild-type hydrolase, wherein the at least one amino acid substitution results in the mutant hydrolase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type hydrolase and the substrate, wherein the at least one amino acid substitution in the mutant hydrolase is a substitution at an amino acid residue in the corresponding wild-type hydrolase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type hydrolase and the substrate or at an amino acid residue in the corresponding wild-type hydrolase that forms an ester intermediate with the substrate, wherein the wild-type hydrolase is a dehalogenase, wherein the mutant hydrolase is a mutant dehalogenase, and wherein the substrate is a compound of formula (I):
- biotin-linker-A—X, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms;
- which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates biotin and X by at least 11 atoms, wherein A—X is a substrate for a dehalogenase, wherein A is (CH2)n and n=2-10, and wherein X is a halogen, and wherein the biotin functional group is coupled through its carboxy terminus to the linker;
- and
- b) detecting or determining the presence or amount of the functional group, thereby detecting or determining the presence or amount of the mutant dehalogenase.
A method of labeling a cell, comprising:
- a) contacting a cell comprising a mutant hydrolase with a hydrolase substrate which comprises one or more biotin functional groups, wherein the mutant hydrolase comprises at least one amino acid substitution relative to a corresponding wild-type hydrolase, wherein the at least one amino acid substitution results in the mutant hydrolase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type hydrolase and the substrate, wherein the at least one amino acid substitution in the mutant hydrolase is a substitution at an amino acid residue in the corresponding wild-type hydrolase that is associated with activating a water molecule which cleaves a bond formed between the corresponding wild-type hydrolase and the substrate or at an amino acid residue in the corresponding wild-type hydrolase that forms an ester intermediate with the substrate, wherein the wild-type hydrolase is a dehalogenase, wherein the mutant hydrolase is a mutant dehalogenase, and wherein the substrate is a compound of formula (I):
- biotin-linker-A—X, wherein the linker is a branched or unbranched carbon chain comprising from 2 to 30 carbon atoms, which chain optionally includes one or more double or triple bonds, and which chain is optionally substituted with one or more hydroxy or oxo (═O) groups, wherein one or more of the carbon atoms in the chain is optionally replaced with a non-peroxide —O—, —S— or —NH—, wherein the linker-A separates biotin and X by at least 11 atoms, wherein A—X is a substrate for a dehalogenase, wherein A is (CH2)n and n=2-10, wherein X is a halogen, and wherein the biotin functional group is coupled through its carboxy terminus to the linker;
- and
- b) detecting or determining the presence or amount of the functional group.
Dependencies are inferred from the claim text.