Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution

Protein-engineering techniques have been adapted for the molecular design o f biosensors that combine a molecular-recognition site with a signal-transd uction function. The optical signal-transduction mechanism of green fluores cent protein (GFP) is most attractive, but hard to combine with a ligand-bi nding site. Here we describe a general method of creating entirely new mole cular-recognition sites on GFPs. At the first step, a protein domain contai ning a desired molecular-binding site is inserted into a surface loop of GF P. Next, the insertional fusion protein is randomly mutated, and new allost eric proteins that undergo changes in fluorescence upon binding of target m olecules are selected from the random library. We have tested this methodol ogy by using TEM1 beta-lactamase and its inhibitory protein as our model pr otein-ligand system, 'Allosteric GFP biosensors' constructed by this method may be used in a wide range of applications including biochemistry and cel l biology. (C) 1999 Federation of European Biochemical Societies.