TY - JOUR
T1 - Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules
AU - Jeon, Hyunjin
AU - Lee, Euiyeon
AU - Kim, Dahee
AU - Lee, Minhyung
AU - Ryu, Jeahee
AU - Kang, Chungwon
AU - Kim, Soyoun
AU - Kwon, Youngeun
N1 - Publisher Copyright:
Copyright © 2018 American Chemical Society.
PY - 2018/8/21
Y1 - 2018/8/21
N2 - Live-cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often use bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from nonspecific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophore-conjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live-cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, dexamethasone. These live-cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.
AB - Live-cell-based biosensors have emerged as a useful tool for biotechnology and chemical biology. Genetically encoded sensor cells often use bimolecular fluorescence complementation or fluorescence resonance energy transfer to build a reporter unit that suffers from nonspecific signal activation at high concentrations. Here, we designed genetically encoded sensor cells that can report the presence of biologically active molecules via fluorescence-translocation based on split intein-mediated conditional protein trans-splicing (PTS) and conditional protein trans-cleavage (PTC) reactions. In this work, the target molecules or the external stimuli activated intein-mediated reactions, which resulted in activation of the fluorophore-conjugated signal peptide. This approach fully valued the bond-making and bond-breaking features of intein-mediated reactions in sensor construction and thus eliminated the interference of false-positive signals resulting from the mere binding of fragmented reporters. We could also avoid the necessity of designing split reporters to refold into active structures upon reconstitution. These live-cell-based sensors were able to detect biologically active signaling molecules, such as Ca2+ and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca2+ stimuli and the glucocorticoid receptor agonist, dexamethasone. These live-cell-based sensing systems hold large potential for applications such as drug screening and toxicology studies, which require functional information about targets.
UR - http://www.scopus.com/inward/record.url?scp=85050621328&partnerID=8YFLogxK
U2 - 10.1021/acs.analchem.8b01481
DO - 10.1021/acs.analchem.8b01481
M3 - Article
C2 - 30028129
AN - SCOPUS:85050621328
SN - 0003-2700
VL - 90
SP - 9779
EP - 9786
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 16
ER -