Highly efficient green fluorescent protein-based kinase substrates.

We have developed a general strategy for designing efficient protein substrates of protein kinases by attaching a phosphorylatable peptide sequence to the C-terminus of His6-tagged green fluorescent protein (GFP). We found that several C-terminal attachment sites in GFP allow for correct presentation of the phosphorylatable tail to a variety of protein kinases. Using this strategy, we have constructed highly efficient GFP-based substrates for Src, c-Abl, protein kinase A, and protein kinase C betaII protein kinases. The engineered GFP substrate for Src (GFP235IYGEFG) is 300 times more efficient than the protein most commonly used as a Src substrate-rabbit muscle enolase.