Destabilization of Green Fluorescent Protein by Substitution of Its Amino-Terminal Residue

Abstract

The Green Fluorescent Protein (GFP) has become a useful tool for molecular biology as a stable reporter system with a half-life greater than 24 hours. However, this stability limits its apphcation in studies which require rapid reporter turnover for example studies of transient kinetics. Here a shortened half-life would be beneficial in accurately measuring the kinetics of transient mRNA transcription from regulated promoters. In previous work Li et al. (Li et al., 1) have created a destabilized protein, pTre-dEGFP+ (t_1/2∼2 h), by a C-terminal fusion of a mouse ornithine decarboxylase (MODC) sequence to EGFP. In our studies we generated destabilized reporter variants by mutations according to the ‘N-end rule’ (Bachmair et al., 2). An essential component of this theory is the role of the proteins N-terminal residue, which determines the proteins half-life. The set of destabilizing amino-terminal residues is organized hierarchically (Bohley, 3), with Arg (R) and Phe (F) generating maximum destabilization.