Method for regulating protein function in cells using synthetic small molecules
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsThomas J. Wandless, Laura Anne Banaszynski, Mari Iwamoto, Lystranne Alysia Maynard, Ling-chun Chen
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Abstract
Methods and compositions for the rapid and reversible destabilizing of specific proteins using cell-permeable, synthetic molecules are described. Stability-affecting proteins, e.g., derived from FKBP and DHFR proteins are fused to a protein of interest and the presence or absence of the ligand is used to modulate the stability of the fusion protein.
Claims25
A conditional protein stability system comprising:
- a nucleic acid sequence encoding a fusion protein that includes
- a protein of interest fused in-frame to
- a single-polypeptide chain, ligand-dependent, stability-affecting protein that is derived from a naturally-occurring ligand binding protein, and
- a ligand that binds to the stability-affecting protein to modulate its stability,
- wherein upon introduction of the nucleic acid sequence to a cell,
- the fusion protein is expressed and
- the stability of the fusion protein can be modulated by amount of ligand present in the cells.
A FKBP variant having one or more amino acid substitutions selected from F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I.
A FKBP variant destabilized by the presence of ligand.
A DHFR variant having one or more amino acid substitutions selected from N18T/A19V, F103L, Y100I, G121V, H12Y/Y100I, H12L/Y100I, R98H/F103S, M42T/H114R, and I61F/T68S.
Cells comprising nucleic acids encoding a fusion protein comprising a protein of interest fused in frame to a variant FKBP or DHFR protein.
Dependencies are inferred from the claim text.