Genetically engineered proteins as recognition receptors

Abstract

The design of fluorescent protein biosensors for the detection of analytes has applications in diverse fields, from molecular life sciences to military readiness. The engineering of biosensors has progressed, from using naturally occurring proteins to the redesign of polypeptide sequences using extensive computational and experimental screening to create entirely new binding molecules. Bacterial periplasmic binding proteins have high affinity and specificity for a variety of biochemically important sugars, amino acids, and anions. The mutation of these proteins and the labeling with fluorescent probes has been utilized to create many reagentless biosensors. Further advances combined genetic fusions of these proteins with derivatives of green fluorescent proteins to create FRET-based biosensors which are expressed in living cells and report on metabolic processes in real-time. A number of proteins and polypeptides have also been used as scaffolds and reengineered to bind new ligands. Computational tools, such as ROSETTA and DEZYMER, have successfully predicted specific mutations to reconstruct protein scaffolds producing novel binders. A complementary approach is to select a highly stable scaffold, randomize predetermined sites, and create phage or ribosome display libraries which may be screened for specificity toward a target analyte. While both of these methods have individually led to the production of novel polypeptides with nanomolar binding constants, the combination of screening libraries and utilizing computational tools is likely to provide the greatest progress in the future of biosensor design. © Springer Science+Business Media, LLC 2010.