Detection of protein translocation by beta-galactosidase reporter fragment complementation

Abstract

Methods and compositions are provided for detecting molecular translocations, particularly protein translocations within and between sub-cellular compartments, using at least two components that exhibit a localization-dependent difference in complementation activity. In particular, alpha-complementing β-galactosidase fragments are provided. These β-galactosidase reporter fragments display significantly enhanced enzymatic activity when one fragment is localized in a membrane. Methods for carrying out no-wash ELISA assays based on the reporter component system are also provided.

What is claimed is: 
     
         1 . A method to assess intracellular translocation of a mammalian protein of interest in a cell, comprising:
 (a) expressing in said cell a first fusion protein of the mammalian protein of interest fused to a β galactosidase peptide alpha fragment having an amino acid sequence according to wild-type SEQ ID NO: 1, provided that said sequence comprises a mutation changing one amino acid in said sequence between residues H31 and E41, or a truncation as shown in SEQ ID NO: 6;   (b) expressing in said cell a second protein fused to a β galactosidase peptide fragment that can complement said alpha fragment to form an active β galactosidase enzyme that generates a detectable signal when said first fusion protein is translocated to allow complexing with said second fusion protein; and   (c) detecting said signal produced by said active complex in said cell to assess intracellular translocation of said mammalian protein of interest.   
     
     
         2 . The method of  claim 1 , wherein the first fusion protein comprises a high affinity fragment of β galactosidase and the intracellular translocation is between subcellular compartments and one of said subcellular compartments is a nucleus. 
     
     
         3 . The method of  claim 1 , wherein the first fusion protein comprises a low affinity fragment of β-galactosidase and the intracellular translocation is between subcellular compartments which are cytosol and a membrane. 
     
     
         4 . The method of  claim 1 , wherein said intracellular translocation is to a nucleus, cytoplasm, or membrane of said cell. 
     
     
         5 . The method of  claim 1 , wherein the complexing is mediated by proximity of said first fusion protein and said second fusion protein. 
     
     
         6 . The method of  claim 1 , wherein the complexing is mediated by binding of said first fusion protein to said second fusion protein. 
     
     
         7 . The method of  claim 8 , wherein the complexing is mediated by affinity of said first fusion protein to said second fusion protein in the presence of a third compound of interest. 
     
     
         8 . The method of  claim 7 , wherein said third compound is a protein. 
     
     
         9 . The method of  claim 1 , wherein said mutation comprises a H31R mutation. 
     
     
         10 . The method of  claim 1 , wherein said first fusion protein comprises is SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 9. 
     
     
         11 . The method of  claim 1 , wherein said second fusion protein comprises SEQ ID NO: 7. 
     
     
         12 . The method of  claim 1 , wherein said second fusion protein comprises a peptide that localizes in a membrane or an in intracellular compartment. 
     
     
         13 . The method of  claim 12 , wherein said peptide is a triplet SV40 nuclear localization signal.