Dataset Information

Development of GFP-based biosensors possessing the binding properties of antibodies.

ABSTRACT: 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.

SUBMITTER: Pavoor TV

PROVIDER: S-EPMC2715507 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Development of GFP-based biosensors possessing the binding properties of antibodies.

Pavoor Tej V TV Cho Yong Ku YK Shusta Eric V EV

Proceedings of the National Academy of Sciences of the United States of America 20090702 29

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 ye ...[more]

PMID: 19574456

Similar Datasets

Project description:The 2018 Nobel Prize in Chemistry recognized in vitro evolution, including the development by George Smith and Gregory Winter of phage display, a technology for engineering the functional capabilities of antibodies into viruses. Such bacteriophages solve inherent problems with antibodies, including their high cost, thermal lability, and propensity to aggregate. While phage display accelerated the discovery of peptide and protein motifs for recognition and binding to proteins in a variety of applications, the development of biosensors using intact phage particles was largely unexplored in the early 2000s. Virus particles, 16.5 MDa in size and assembled from thousands of proteins, could not simply be substituted for antibodies in any existing biosensor architectures.Incorporating viruses into biosensors required us to answer several questions: What process will allow the incorporation of viruses into a functional bioaffinity layer? How can the binding of a protein disease marker to a virus particle be electrically transduced to produce a signal? Will the variable salt concentration of a bodily fluid interfere with electrical transduction? A completely new biosensor architecture and a new scheme for electrical transduction of the binding of molecules to viruses were required.This Account describes the highlights of a research program launched in 2006 that answered these questions. These efforts culminated in 2018 in the invention of a biosensor specifically designed to interface with virus particles: the Virus BioResistor (VBR). The VBR is a resistor consisting of a conductive polymer matrix in which M13 virus particles are entrained. The electrical impedance of this resistor, measured across 4 orders of magnitude in frequency, simultaneously measures the concentration of a target protein and the ionic conductivity of the medium in which the resistor is immersed. Large signal amplitudes coupled with the inherent simplicity of the VBR sensor design result in high signal-to-noise ratio (S/N > 100) and excellent sensor-to-sensor reproducibility. Using this new device, we have measured the urinary bladder cancer biomarker nucleic acid deglycase (DJ-1) in urine samples. This optimized VBR is characterized by extremely low sensor-to-sensor coefficients of variation in the range of 3-7% across the DJ-1 binding curve down to a limit of quantitation of 30 pM, encompassing 4 orders of magnitude in concentration.

Dataset Information

Development of GFP-based biosensors possessing the binding properties of antibodies.

Publications

Development of GFP-based biosensors possessing the binding properties of antibodies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets