Reagents and systems for generating biosensors
AssigneesThe Regents of the University of California, The Regents of the University of Colorado, a body corporate
InventorSean R. Cutler
Life of this patent
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Abstract
The disclosure relates to reagents and systems for generating a biosensor employing plant abscisic acid receptor polypeptide mutagenized in the ligand-binding pocket as a scaffold to detect small molecules. Here, we describe an approach for the rapid engineering of biosensors using a PYR1/PYL receptor polypeptide, a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and'a requirement for ligand-induced heterodimerization, which facilitates the construction of sense-response functions.
Claims27
A library comprising a plurality of polypeptides, wherein each polypeptide contained in the plurality of polypeptides comprises a mutated PYR/PYL receptor polypeptide comprising I, L, R, V, or Y at position 62, relative to the corresponding position of SEQ ID NO:1;
- and at least two amino acid substitutions, relative to the corresponding position of SEQ ID NO:
- 1, selected from positions 59, 83, 89, 92, 94, 108, 117, 120, 122, 141, 159, 160, and 163, wherein the at least two amino acid substitutions differ among members of the library and are independently selected from A, D, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W and Y.
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-26. (canceled)
A HAB1 polypeptide comprising an N-terminus deletion, wherein the HAB1 polypeptide has at least 95% identity to SEQ ID NO:3 and comprises the amino acid residues at positions 393, 406, 411, 416, 455, 467, 481, 494, 499, 549, 565, 584, 629, 646, and 698 as set forth in SEQ ID NO:3;
- or comprises the amino acid sequence of SEQ ID NO:3.
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A cannabinoid-binding polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOS:
- 6-22, wherein the polypeptide comprises each of the highlighted amino acid residues in the corresponding sequence;
- or comprises the amino acid sequence of any one of SEQ ID NOS:
- 6-22;
- or
- an organophosphate-binding polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOS:
- 23-30, wherein the polypeptide comprises each of the highlighted amino acid residues in the corresponding sequence;
- or the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:
- 23-30;
- or
- a cannabinoid and organophosphate-binding polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO:31 or 32, wherein the polypeptide comprises each of the highlighted amino acid residues in the corresponding sequence;
- or the polypeptide comprises the amino acid sequence of SEQ ID NO:31 or 32;
- or
- a biosensor polypeptide comprising an amino acid sequence having at least 95% identity to any one of SEQ ID NOS:
- 33-37, wherein the polypeptide comprises each of the bolded amino acid residues in the corresponding sequence;
- or the polypeptide comprises the amino acid sequence of any one of SEQ ID NOS:
- 33-37.
-38. (canceled)
Dependencies are inferred from the claim text.