Light-inducible system for regulating protein stability
Disclosed herein are a light-inducible system and method for rapidly and reversibly modulating protein stability and function. This system and method employs conditionally stable protein domains that regulate the degradation of a fusion protein depending upon the presence or absence of a particular light source.
1. A light-regulated conditional protein stability system, comprising:
a nucleic acid sequence encoding a fusion protein comprising a protein of interest fused in-frame to a light-regulated, stability-affecting polypeptide comprised of a light-sensitive degradation domain and a degron peptide selected from the group consisting of peptides identified as SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 10,
wherein, upon introduction of the nucleic acid sequence to a cell, the fusion protein is expressed and stability of the fusion protein is modulated by exposure of the cell to a non-toxic light.
2. The system of claim 1 , wherein the light-regulated, stability-affecting polypeptide comprises an amino acid sequence selected from the group of sequences identified by SEQ ID NOs: 3-8.
3. The system of claim 1 , wherein the cell is stably transformed with the nucleic acid.
4. The system of claim 3 , wherein the stably transformed cell is implanted in a living animal.
5. The system of claim 1 , wherein the nucleic acid sequence is in a viral vector.
6. The system of claim 5 , wherein the viral vector is a vaccinia virus.
7. The system of claim 1 , wherein the non-toxic light has a wavelength of approximately 450-470 nm.
8. A light sensitive degradation domain (DD) comprising an amino acid sequence selected from the group of sequences identified by SEQ ID NOs: 3-8.
9. A fusion protein comprising the light sensitive DD of claim 8 .
10. A cell comprising a nucleic acid encoding a fusion protein comprising a protein of interest fused in-frame to a light-regulated, stability-affecting polypeptide, wherein the light-regulated stability-affecting polypeptide is a polypeptide having 95% sequence identity to SEQ ID NO: 4, and wherein the light-regulated stability-affecting polypeptide is fused at its C-terminus to a degradation domain selected from the sequences identified as SEQ ID NO: 2 and SEQ ID NO: 9.
11. A kit of parts comprising the system of claim 1 along with instructions for use.
12. The system of claim 1 , wherein the light-regulated, stability-affecting polypeptide is selected from (i) SEQ ID NO: 3 and (ii) sequences having 95% sequence identity to SEQ ID NO: 3.
13. The system of claim 1 , wherein the light-regulated, stability-affecting polypeptide is selected from (i) SEQ ID NO: 4 and (ii) sequences having 95% sequence identity to SEQ ID NO: 4.
14. The light sensitive degradation domain of claim 8 , wherein the light sensitive degradation domain is SEQ ID NO: 3.
15. A fusion protein comprising the light sensitive degradation domain of claim 14 .
16. The system of claim 1 , wherein the light-regulated stability-affecting polypeptide is a polypeptide having 95% sequence identity to SEQ ID NO: 4 fused at its C-terminus to a degradation domain selected from the sequences identified as SEQ ID NO: 2 and SEQ ID NO: 9.
17. The system of claim 1 , wherein the light-regulated stability-affecting polypeptide is a sequence with 98% identity to a sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, the sequence fused at its C-terminus to a degradation domain selected from the sequences identified as SEQ ID NO: 2 and SEQ ID NO: 9.