Background: Site-selective modification of proteins at two separate locations using two different reagents is highly desirable for biosensor applications employing fluorescence resonance energy transfer (FRET), but few strategies are available for such modification. To address this challenge, sequential selective modification of two cysteines in glucose/galactose binding protein (GGBP) was demonstrated using a technique we call "ligand protection."
Method: In this technique, two cysteines were introduced in GGBP and one cysteine is rendered inaccessible by the presence of glucose, thus allowing sequential attachment of two different thiol-reactive reagents.
Periplasmic expression screening is a selection technique used to enrich high-affinity proteins in Escherichia coli. We report using this screening method to rapidly select a mutated D-glucose/D-galactose-binding protein (GGBP) having low affinity to glucose. Wild-type GGBP has an equilibrium dissociation constant of 0.
View Article and Find Full Text PDFEnvironmentally sensitive near-IR (NIR) dyes are useful fluorophores for various biosensor applications when tissue absorption, scattering, and autofluorescence are a leading concern. Biosensors operating in the NIR region (generally wavelengths >650 nm) would avoid interference from biological media and thereby facilitate relatively interference free sensing. Squaraine dyes are potential candidates to serve as reporter molecules due to their spectral properties in the NIR region, but none is commercially available for site-specific coupling to proteins through native or engineered thiols on cysteine.
View Article and Find Full Text PDFBackground: Fluorescent biosensors based on galactose/glucose binding protein (GGBP) and environmentally sensitive derivatives of the phenoxazine dye Nile Red are described. These biosensors are proposed as the sensing platform for a minimally invasive, continuous glucose monitoring system that can be implanted under the skin and read transdermally using an external fluorometer.
Methods: To construct the biosensors, the thiol-reactive Nile Red derivatives INR and IANR were prepared and conjugated to GGBP proteins possessing cysteine mutations that were designed for optimal site-specific fluorophore attachment.
The monitoring and management of blood glucose levels are key components for maintaining the health of people with diabetes. Traditionally, glucose monitoring has been based on indirect detection using electrochemistry and enzymes such as glucose oxidase or glucose dehydrogenase. Here, we demonstrate direct detection of glucose using a surface plasmon resonance (SPR) biosensor.
View Article and Find Full Text PDFAdeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site.
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