Development of GFP-based biosensors possessing the binding properties of antibodies.
Proteins that can bind specifically to targets that also have an intrinsic property allowing for easy detection could facilitate a multitude of applications. While the widely used green fluorescent protein (GFP) allows for easy detection, attempts to insert multiple binding loops into GFP to impart affinity for a specific target have been met with limited success because of the structural sensitivity of the GFP chromophore. In this study, directed evolution using a surrogate loop approach and yeast surface display yielded a family of GFP scaffolds capable of accommodating 2 proximal, randomized binding loops. The library of potential GFP-based binders or ''GFAbs'' was subsequently mined for GFAbs capable of binding to protein targets. Identified GFAbs bound with nanomolar affinity and required binding contributions from both loops indicating the advantage of a dual loop GFAb platform. Finally, GFAbs were solubly produced and used as fluorescence detection reagents to demonstrate their utility.
Pavoor
Tej V
TV
Department of Chemical and Biological Engineering, University of Wisconsin, Madison, WI 53706, USA.
Cho
Yong Ku
YK
Shusta
Eric V
EV
D000906:Antibodies / Q000302:isolation & purification / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D002999:Clone Cells; D019020:Directed Molecular Evolution; D005453:Fluorescence; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017433:Protein Structure, Secondary; D012441:Saccharomyces cerevisiae; D012995:Solubility
D000906:Antibodies; D050505:Mutant Proteins; D049452:Green Fluorescent Proteins