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

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Abstract
Protein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 β-lactamase and its inhibitory protein as our model protein-ligand system. ‘Allosteric GFP biosensors’ constructed by this method may be used in a wide range of applications including biochemistry and cell biology.