DETECTION OF PROTEIN TRANSLOCATION BY BETA-GALACTOSIDASE REPORTER FRAGMENT COMPLEMENTATION

摘要

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.

主权项

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.