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 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 beta-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.
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo, Japan.
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D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D019020:Directed Molecular Evolution / Q000379:methods*; D005453:Fluorescence; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D016254:Mutagenesis, Insertional; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D015398:Signal Transduction; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D008024:Ligands; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D065093:beta-Lactamase Inhibitors; C065596:beta-lactamase-inhibitor protein, Streptomyces; D049452:Green Fluorescent Proteins; D001618:beta-Lactamases