Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules
| Autor: | Hyunjin Jeon, Youngeun Kwon, Dahee Kim, Minhyung Lee, Euiyeon Lee, Jeahee Ryu, Soyoun Kim, Chungwon Kang |
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| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Cell signaling Chemical biology Biosensing Techniques Protein Sorting Signals Protein Engineering 010402 general chemistry 01 natural sciences Inteins Analytical Chemistry 03 medical and health sciences Bimolecular fluorescence complementation Calmodulin Protein splicing Humans Protein Splicing Amino Acid Sequence Cell Engineering Chemistry Protein engineering Hormones 0104 chemical sciences Luminescent Proteins 030104 developmental biology Förster resonance energy transfer Exteins Biophysics Calcium Intein Biosensor HeLa Cells |
| Zdroj: | Analytical Chemistry. 90:9779 |
| ISSN: | 1520-6882 0003-2700 |
| DOI: | 10.1021/acs.analchem.8b01481 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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