Rapidly degrading GFP-fusion proteins and methods of use

Abstract

Green fluorescent protein (GFP) is widely used as a reporter in determining gene expression and protein localization. The present invention provides fusion proteins with a half life of ten hours or less with several embodiments having half lives of 4 hours or less. Such proteins may be constructed by fusing C-terminal amino acids of the degradation domain of mouse ornithine decarboxylase (MODC), which contains a PEST sequence, to the C-terminal end of an enhanced variant of GFP (EGFP). Fluorescence intensity of the fusion protein in transfected cells is similar to that of EGFP, but the fusion protein, unlike EGFP, is unstable in the presence of cycloheximide. Specific mutations in the MODC region have resulted in mutants with varying half lives, useful for a variety of purposes.

1 - 17 . (canceled)  
     
     
         18 . A method of assaying activation or deactivation of transcriptional or translational elements with a transient fluorescent reporter protein, comprising the steps of: 
 transfecting cells with an expression vector comprising a fluorescent protein fusion protein having a half life of no more than about ten hours, wherein the fusion protein is under the influence of the promoter, transcriptional or translational element; and    detecting the presence, absence or amount of fluorescence in said cells.    
     
     
         19 . The method of  claim 18 , wherein the amount of fluorescence present in the cell is a measure of the fluorescent protein that is being expressed.  
     
     
         20 . A method of assaying activation or deactivation of promoters or other transcriptional or translational elements with a transient fluorescent protein reporter protein, comprising the steps of: 
 transfecting cells with an expression vector comprising a fluorescent fusion protein having a half life of no more than about ten hours, wherein the fluorescent fusion protein is under an influence of said promoter, transcriptional or translational element;    treating said transfected cells with a compound of interest; and    detecting a change in fluorescence upon treatment of the cells with said compound of interest so as to assay the effect of said compound of interest on said activation or deactivation of said transcription or translation elements.    
     
     
         21 . A method of studying cell lineage, comprising the steps of: 
 transfecting undifferentiated cells with a vector expressing the destabilized fusion protein of claim  1 ;    growing said undifferentiated cells under conditions in which the undifferentiated cells become differentiated cells; and    detecting an absence, presence or location of fluorescence in the differentiated cells.    
     
     
         22 . A method of using a fusion protein of claim  1  in cell localization studies, comprising the steps of: 
 transfecting cells with an expression vector comprising a GFP fusion protein having a half life of no more than ten hours, wherein the fusion protein is linked to a putative cell localization element; 
 growing the cell; and  
   detecting a location of fluorescence in the cells.