Covalent tethering of functional groups to proteins

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.

1. 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 (CH 2 ) 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. 
     
     
       2. 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 (CH 2 ) n  and n=2-10, and wherein X is a halogen. 
     
     
       3. 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 (CH 2 ) 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. 
     
     
       4. The compound of  claim 3  which is a substrate for a Rhodococcus dehalogenase. 
     
     
       5. The compound of  claim 3  wherein X is Cl or Br. 
     
     
       6. The compound of  claim 3  wherein the linker comprises 3 to 30 atoms. 
     
     
       7. The compound of  claim 3  wherein the linker has 11 to 30 atoms. 
     
     
       8. The compound of  claim 3  which is N-{2-[2-(6-Chlorohexyloxy)-ethoxy]-ethyl}-biotin-amide. 
     
     
       9. The compound of  claim 3  wherein biotin is separated from A—X by up to 100 angstroms. 
     
     
       10. The compound of  claim 3  wherein biotin is separated from A—X by up to 500 angstroms. 
     
     
       11. The compound of  claim 3  wherein the chain comprises (CH 2 CH 2 O) y  and y=2-8. 
     
     
       12. A compound prepared by the method of  claim 1  wherein the compound is: 
       
         
           
           
               
               
           
         
       
       
        
       
     
     
       13. 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 (CH 2 ) 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. 
     
     
       14. A compound of formula (II): 
       
         
           
           
               
               
           
         
       
     
     
       15. A compound prepared by the method of  claim 1 . 
     
     
       16. 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 (CH 2 ) n  and n=2-10 n=6-10, 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 biotin, thereby detecting or determining the presence or amount of the mutant dehalogenase. 
     
     
       17. The method of  claim 16  wherein the substitution is at a residue in the wild-type dehalogenase that activates the water molecule. 
     
     
       18. The method of  claim 17  wherein the residue in the wild-type dehalogenase that activates the water molecule is histidine. 
     
     
       19. The method of  claim 16  wherein the substitution is at a residue in the wild-type dehalogenase that forms an ester intermediate with the substrate. 
     
     
       20. The method of  claim 19  wherein the residue in the wild-type dehalogenase that forms an ester intermediate with the substrate is aspartate. 
     
     
       21. 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 (CH 2 ) n  and n=2-10 n=6-10, 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. 
     
     
       22. The method of  claim 21  wherein the substitution is at a residue in the wild-type dehalogenase that activates the water molecule. 
     
     
       23. The method of  claim 21  wherein the residue in the wild-type dehalogenase that activates the water molecule is histidine. 
     
     
       24. The method of  claim 21  wherein the substitution is at a residue in the wild-type dehalogenase that forms an ester intermediate with the substrate. 
     
     
       25. The method of  claim 24  wherein the residue in the wild-type dehalogenase that forms an ester intermediate with the substrate is aspartate. 
     
     
       26. The method of  claim 16  or  21  wherein the linker comprises (CH 2 CH 2 ) y  and y=2-8. 
     
     
       27. The method of  claim 16  or  21  wherein the linker separates biotin and A by at least 12 atoms. 
     
     
       28. The method of any one of  claim 16  or  21  wherein the mutant dehalogenase is present in a cell or on the surface of a cell. 
     
     
       29. The method of any one of  claims 16  or  21  wherein the presence of at least one biotin functional group in a cell is correlated to the subcellular location of the mutant dehalogenase. 
     
     
       30. The method of any one of  claim 16  or  21  wherein the mutant dehalogenase forms a fusion protein with a protein of interest. 
     
     
       31. The method of  claim 30  wherein the protein of interest is a selectable marker protein, membrane protein, cytosolic protein, nuclear protein, structural protein, an enzyme, an enzyme substrate, a receptor protein, a transporter protein, a transcription factor, a channel protein, a phospho-protein, a kinase, a signaling protein, a metabolic protein, a mitochondrial protein, a receptor associated protein, a nucleic acid binding protein, an extracellular matrix protein, a secreted protein, a receptor ligand, a serum protein, an immunogenic protein, a fluorescent protein, or a protein with reactive cysteine. 
     
     
       32. The method of  claim 21  wherein the mutant dehalogenase further comprises a selectable marker protein. 
     
     
       33. The method of  claim 32  wherein the mutant dehalogenase forms an ester bond with the substrate. 
     
     
       34. The method of  claim 32  wherein the mutant dehalogenase forms a thioester bond with the substrate. 
     
     
       35. The method of  claim 21  further comprising contacting the cell with a fixative prior to or after contacting the cell with the substrate. 
     
     
       36. The method of  claim 21  further comprising contacting the cell with a fixative concurrently with contacting the cell with the substrate. 
     
     
       37. The method of  claim 35  or  36  wherein the cell is fixed with methanol, acetone and/or paraformaldehyde. 
     
     
       38. The method of  claim 32  further comprising contacting the cell with a fixative prior to or after contacting the cell with the substrate. 
     
     
       39. The method of  claim 32  further comprising contacting the cell with a fixative concurrently contacting the cell with the substrate. 
     
     
       40. The method of  claim 38  or  39  wherein the cell is fixed with methanol, acetone and/or paraformaldehyde.