System for protease mediated protein expression
View Patent ↗The present invention provides methods and materials that can be used to detect, examine and exploit protease mediated protein expression. An illustrative embodiment of the invention is a polynucleotide encoding a polypeptide having a plurality of operatively linked elements comprising a polypeptide degradation signal that functions as a proteosome substrate; a polypeptide of interest having an observable activity; and a linker disposed between the polypeptide degradation signal and the polypeptide of interest. The linker comprises a protease recognition motif that is capable of being specifically recognized and cleaved by a protease. The activity of the polypeptide of interest is observed when the motif is specifically recognized and cleaved by the protease; and the activity of the polypeptide of interest is decreased or not observed when the motif is not specifically recognized and cleaved by the protease.
1. A polynucleotide encoding a protease-regulated polypeptide comprising the following operatively linked elements:
(a) a nucleic acid sequence encoding a protein degradation signal;
(b) a nucleic acid sequence encoding a polypeptide of interest; and
(c) a nucleic acid sequence encoding a linker set forth as SEQ ID NO:1 disposed between (a) and (b), wherein the linker comprises a protease recognition motif that is cleaved by a protease;
wherein, in the presence of the protease, cleavage of the linker protects the polypeptide of interest from proteasomal degradation; and, in the absence of the protease, activity of the polypeptide of interest is decreased.
2. A first vector comprising the polynucleotide of claim 1 .
3. A cell comprising the first vector of claim 2 .
4. The polynucleotide of claim 1 , wherein the protein degradation signal is a ubiquitin degradation signal.
5. The polynucleotide of claim 1 , wherein the polypeptide of interest is a reporter protein whose expression can be detected.
6. The polynucleotide of claim 1 , wherein the polypeptide of interest is a therapeutically active polypeptide.
7. The first vector of claim 2 , further comprising a promoter and/or an enhancer that modulates the expression of the protease-regulated polypeptide.
8. The cell of claim 3 , wherein the polypeptide of interest is a first reporter protein, and the cell further comprises a second vector that encodes a second reporter protein whose expression is modulated by a tissue specific promoter or a tissue specific enhancer.
9. A kit comprising the polynucleotide of claim 1 and a suitable container.