2 research outputs found
Flashbody: A Next Generation Fluobody with Fluorescence Intensity Enhanced by Antigen Binding
Fluorescent
probes are valuable tools for visualizing the spatiotemporal dynamics
of molecules in living cells. Here we developed a genetically encoded
antibody probe with antigen-dependent fluorescence intensity called
“Flashbody”. We first created a fusion of EGFP to the
single chain variable region fragment (scFv) of antibody against seven
amino acids of the bone Gla protein C-terminus (BGPC7) called BGP
Fluobody, which successfully showed the intracellular localization
of BGPC7-tagged protein. To generate BGP Flashbody, circularly permuted
GFP was inserted in between two variable region fragments, and the
linkers were optimized, resulting in fluorescence intensity increase
of 300% upon binding with BGPC7 in a dose-dependent manner. Live-cell
imaging using BGP Flashbody showed that BGPC7 fused with cell penetrating
peptide was able to enter through the plasma membrane by forming a
nucleation zone, while it penetrated the nuclear membrane with different
mechanism. The construction of Flashbody will be possible for a range
of antibody fragments and opens up new possibilities for visualizing
a myriad of molecules of interest
Generation of a cGMP Indicator with an Expanded Dynamic Range by Optimization of Amino Acid Linkers between a Fluorescent Protein and PDE5α
Here
we describe the development of a single fluorescent protein
(FP)-based cGMP indicator, Green cGull, based on the cGMP binding
domain from mouse phosphodiesterase 5α. The dynamic range of
Green cGull was enhanced to a 7.5-fold fluorescence change upon cGMP
binding by optimization of the amino acid linkers between the cGMP
binding domain and FP. Green cGull has excitation and emission peaks
at 498 and 522 nm, respectively, and specifically responds to cGMP
in a dose-dependent manner. Live cell imaging analysis revealed that
addition of a nitric oxide (NO) donor induced different cGMP kinetics
and was cell-type dependent. We also found that the NO donor induced
an increase of intracellular cGMP, while intracellular Ca<sup>2+</sup> exhibited a complex profile, as revealed by dual-color imaging of
cGMP and Ca<sup>2+</sup>. The results suggest that Green cGull sheds
new light on understanding the complex interactions between various
signaling molecules by multicolor imaging and that our systematic
strategy for expanding the dynamic range of single-FP-based indicators
is valuable to generate indicators for molecules of interest