Sensor proteins and assay methods
View Patent ↗The present invention relates to biosensors. In some embodiments, the biosensors are modified ligand binding molecules. In some embodiments, the modified ligand binding molecule is a phosphate binding protein (PBP). In some embodiments, the modified ligand binding molecules are labeled to be capable of RET, e.g., comprising a donor and acceptor moiety. In some embodiments of the invention, there is a detectable change in RET (e.g., FRET) when the modified ligand binding molecule binds and/or releases the ligand (e.g., phosphate). The invention also provides related methods, reactions and assays.
1. A phosphate binding protein comprising a resonance energy transfer (RET) pair of moieties comprised of at least one donor moiety and at least one acceptor moiety, wherein the donor moiety comprises a lanthanide metal complex, wherein neither the donor nor acceptor moiety is a fluorescent protein, and wherein the phosphate binding protein is capable of binding inorganic phosphate and wherein the binding results in a change in RET.
2. The protein of claim 1 , wherein the distance or orientation between the at least two moieties is altered upon binding the phosphate.
3. The protein of claim 1 , wherein the RET pair is capable of time resolved RET.
4. The protein of claim 1 , wherein the at least one acceptor moiety is selected from the group consisting of a fluorescein, a rhodamine, a FITC, a 5-carboxyfluorescein, a 6-carboxyfluorescein, a 7-hydroxycoumarin-3-carboxamide, a 6-chloro-7-hydroxycoumarin-3-carboxamide, a fluorescein-5-isothiocyanate, a dichlorotriazinylaminofluorescein, a tetramethylrhodamine-5-isothiocyanate, tetramethylrhodamine-6-isothiocyanate, a succinimidyl ester of 5-carboxyfluorescein, a succinimidyl ester of 6-carboxyfluorescein, a 5-carboxytetramethylrhodamine, a 6-carboxymethylrhodamine, a 7-amino-4-methylcoumarin-3-acetic acid, 6-IAF, 5-IAF, fluorescein-5-maleimide, and 5-(bromomethyl)fluorescein.
5. The protein of claim 1 , wherein the lanthanide metal complex comprises an organic antenna moiety, a metal liganding moiety and a lanthanide metal ion.
6. The protein of claim 5 , wherein the lanthanide metal ion is selected from the group consisting of: Sm(III), Ru(III), Eu (III), Gd(III), Tb(III), and Dy(III).
7. The protein of claim 5 , wherein the organic antenna moiety is selected from the group consisting of: rhodamine 560, fluorescein 575, fluorescein 590, 2-quinolone, 4-quinolone, 4-trifluoromethylcoumarin (TFC), 7-diethyl-amino-coumarin-3-carbohydrazide, 7-amino-4-methyl-2-coumarin (carbostyril 124), 7-amino-4-methyl-2-coumarin (coumarin 120), 7-amino-4-trifluoromethyl-2-coumarin (coumarin 124), and aminomethyltrimethylpsoralen.
8. The protein of claim 5 , wherein the metal liganding moiety is a metal chelating moiety selected from the group consisting of: EDTA, DTPA, TTHA, DOTA, NTA, HDTA, DTPP, EDTP, HDTP, NTP, DOTP, DO3A, DOTAGA, and NOTA.
9. The protein of claim 1 , wherein the lanthanide metal complex has a structure:
-L n -A-S n -C M ,
or
-L n -C M -S n -A,
wherein A represents an organic antenna moiety;
L represents a linker;
S represents a spacer;
n can be 0 or 1;
C represents a metal chelating moiety; and
M represents a lanthanide metal ion coordinated to C.
10. The protein of claim 1 , wherein the protein has at least one non-native cysteine amino acid and the first or second moiety is attached to the non-native cysteine amino acid.
11. The protein of claim 10 , wherein the protein comprises a modified amino acid sequence of the phosphate binding protein encoded by the phoS gene.
12. The protein of claim 1 , wherein the amino acid sequence of the protein is SEQ ID NO:1 or SEQ ID NO:2.
13. The protein of claim 1 , wherein the phosphate binding protein comprises an amino acid sequence 90% homologous to SEQ ID NO:1 or SEQ ID NO:2.
14. The protein of claim 13 , wherein the protein has at least one non-native cysteine amino acid.
15. The protein of claim 13 , comprising an amino acid substitution selected from the group consisting of A47C, A197C, Q201C and E268C of SEQ ID NO:2.
16. The protein of claim 1 , wherein the at least one donor moiety is linked to the phosphate binding protein via an amine or thiol linkage.
17. The protein of claim 1 , wherein the at least one acceptor moiety is linked to the phosphate binding protein via an amine or thiol linkage.
18. The protein of claim 13 , comprising at least 2 amino acid substitutions selected from the group consisting of A197C/E268C, A47C/A197C, A47C/E268C, Q201C/E268C, A47C/Q201C, and A197C/Q201C of SEQ ID NO:2.
19. The protein of claim 12 , wherein the at least one donor moiety or the at least one acceptor moiety is linked to the protein at a region selected from the group consisting of amino acids 42-51 of SEQ ID NO:2, amino acids 149-159 of SEQ ID NO:2, amino acids 180-191 of SEQ ID NO:2, amino acids 192-201 of SEQ ID NO:2 and amino acids 264-273 of SEQ ID NO:2.
20. The protein of claim 13 , wherein the at least one donor moiety or the at least one acceptor moiety is linked to the protein at a region selected from the group consisting of amino acids 42-51 of SEQ ID NO:2, amino acids 149-159 of SEQ ID NO:2, amino acids 180-191 of SEQ ID NO:2, amino acids 192-201 of SEQ ID NO:2 and amino acids 264-273 of SEQ ID NO:2.