Fusion proteins for identifying proteases, protease target sites and regulators of protease activity in living cells
The invention provides a fusion protein including a reporter polypeptide, a linker polypeptide comprising a protease cleavage site, and a repressor polypeptide. The repressor polypeptide represses the activity of the reporter polypeptide by conferring a specific localization in a cell that reduces activity of the reporter activity until the cleavage site is cleaved. A method is also provided for identifying a protease that recognizes a specific protease cleavage site. The invention further provides a method of identifying a compound that activates a protease.
1. A fusion protein comprising:
a) a reporter polypeptide comprising a C-terminal Lex A-B42 transcription factor linked to a linker polypeptide comprising a protease cleavage site; and
b) a repressor polypeptide comprising an N-terminal fragment of CD4 that represses transcriptional activity of the reporter polypeptide by conferring a specific localization in a cell such that the reporter polypeptide has reduced transcriptional activity, wherein said repressor polypeptide is linked to the linker polypeptide, and
wherein, upon cleavage of said linker polypeptide at said protease cleavage site, an increase in the transcriptional activity of said reporter polypeptide can be detected.
2. The fusion protein of claim 1 , wherein said protease cleavage site is a caspase cleavage site.
3. A fusion protein comprising:
a) a reporter polypeptide comprising a C-terminal Lex A-B42 transcription factor linked to a linker polypeptide comprising a protease cleavage site; and
b) a repressor polypeptide comprising an N-terminal fragment of CD4 or an amyloid precursor protein that represses transcriptional activity of the reporter polypeptide by conferring a specific localization in a cell such that the reporter polypeptide has reduced tscriptional activity, wherein said repressor polypeptide is linked to the linker polypeptide, and
wherein, upon cleavage of said linker polypeptide at said protease cleavage site, an increase in the transcriptional activity of said reporter polypeptide can be detected.
4. The fusion protein of claim 3 , wherein said protease cleavage site is a caspase cleavage site.