Tags

Type your tag names separated by a space and hit enter

Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules.
Anal Chem. 2018 08 21; 90(16):9779-9786.AC

Abstract

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.

Authors+Show Affiliations

Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.Department of Biomedical Engineering (BK21 plus) , Dongguk University , Seoul 04620 , Korea.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30028129

Citation

Jeon, Hyunjin, et al. "Cell-Based Biosensors Based On Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules." Analytical Chemistry, vol. 90, no. 16, 2018, pp. 9779-9786.
Jeon H, Lee E, Kim D, et al. Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules. Anal Chem. 2018;90(16):9779-9786.
Jeon, H., Lee, E., Kim, D., Lee, M., Ryu, J., Kang, C., Kim, S., & Kwon, Y. (2018). Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules. Analytical Chemistry, 90(16), 9779-9786. https://doi.org/10.1021/acs.analchem.8b01481
Jeon H, et al. Cell-Based Biosensors Based On Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules. Anal Chem. 2018 08 21;90(16):9779-9786. PubMed PMID: 30028129.
* Article titles in AMA citation format should be in sentence-case
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, Y1 - 2018/08/03/ PY - 2018/7/22/pubmed PY - 2019/4/9/medline PY - 2018/7/21/entrez SP - 9779 EP - 9786 JF - Analytical chemistry JO - Anal. Chem. VL - 90 IS - 16 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. SN - 1520-6882 UR - https://www.unboundmedicine.com/medline/citation/30028129/Cell_Based_Biosensors_Based_on_Intein_Mediated_Protein_Engineering_for_Detection_of_Biologically_Active_Signaling_Molecules_ L2 - https://doi.org/10.1021/acs.analchem.8b01481 DB - PRIME DP - Unbound Medicine ER -