| 主权项 |
1. A method for conditionally stabilizing a protein of interest (POI), comprising:
fusing a nucleic acid encoding the POI in-frame to a nucleic acid encoding a FK506-binding protein (FKBP) variant or dihydrofolate reductase (DHFR) variant to produce a nucleic acid encoding a fusion protein comprising the FKBP variant or DHFR variant, introducing the nucleic acid encoding the fusion protein into a cell, expressing the fusion protein in the cell, and (i) conditionally stabilizing the fusion protein in the presence of a ligand that binds:
(a) the FKBP variant, wherein the FKBP variant has an F36V amino acid substitution and one or more amino acid substitutions selected from F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I, wherein the FKBP variant destabilizes the POI in the absence of the ligand and the FKBP variant stabilizes the POI in the presence of the ligand, and wherein the ligand that binds the FKBP variant is Shield1, FK506 or SLF*; or(b) the DHFR variant, wherein the DHFR variant has one or more amino acid substitutions selected from the group consisting of N18T/A19V, F103L, Y100I, G121V, H12Y/Y100I, H12L/Y100I, R98H/F103S, M42T/H114R, and I61F/T68S, wherein the DHFR variant destabilizes the POI in the absence of the ligand and stabilizes the POI in the presence of the ligand, and wherein the ligand that binds the DHFR variant is trimethoprim (TMP); or (ii) conditionally stabilizing the fusion protein in the absence of a ligand that binds the FKBP variant, wherein the fusion protein comprises the FKBP variant identified as SEQ ID NO:1 and the peptide identified as SEQ ID NO:18, wherein the N-terminus of the peptide identified as SEQ ID NO:18 is fused to the C-terminus of the FKBP variant identified as SEQ ID NO:1, wherein the FKBP variant stabilizes the POI in the absence of an FKBP binding ligand, wherein the ligand that binds the FKBP variant is Shield1, FK506 or SLF*. |
