Cell-Based Biosensors Based on Intein-Mediated Protein Engineering for Detection of Biologically Active Signaling Molecules

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 Ca and cortisol, as well as relevant biological stimuli, such as histamine-induced Ca 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.

Rapid Screening of Glucocorticoid Receptor (GR) Effectors Using Cortisol-Detecting Sensor Cells

“…The signal peptides translocated the fluorescent cargo from the nucleus to cytosol to report the presence of target molecules ( Scheme 2 ). For the screening of natural products, we engineered sensor cells, equipped with accelerated reporting capabilities that instantaneously activated after covalent bond formation, as the long response time severely limited the practical use of the sensor cells [ 29 ]. We evaluated the performance of the developed sensors and used them for rapid screening of cortisol analogues in various essential oils often used for stress relief.…”

Genetically Encoded Sensor Cells for the Screening of Glucocorticoid Receptor (GR) Effectors in Herbal Extracts

“…In order to overcome the limitations of BiFC and FRET-based approaches, we have previously designed a reporting system based on an intein-mediated reconstitution of the signal peptide [ 17 , 18 , 19 ]. Complemented split-inteins self-catalytically react to form and break specific peptide bonds without requiring an energy source or cofactors [ 20 , 21 ].…”

“…Split-signal peptides are instantly activated through covalent bond formation without a tedious refolding step and cannot be activated by merely binding the split-fragments; therefore, this approach can address the limitations of BiFC and FRET-based reporting systems. These reporter systems have been designed into cell-based sensors that can detect various bioactive targets, including Ca 2+ , cortisol, and rapamycin [ 17 , 18 , 19 ]. These sensor cells demonstrated excellent performance in screening targets with enhanced sensitivity with a short response time.…”

Proximity Induced Splicing Utilizing Caged Split Inteins

“…To address this issue, a variety of conditional protein splicing (CPS) methods have been developed in which intein activity is activated only upon addition of a specified trigger. Generally, these triggers induce splicing by introducing either a splicing favorable conformational change in an otherwise structurally locked intein, − deprotecting chemically caged residues that inhibit catalytic activity − or bringing intein fragments with low inherent affinity into close proximity. ,− …”

“…An appealing feature of this CPS strategy is the potential to exchange the triggering mechanism. ,− As such, we were especially interested in the idea of using DNA as a substrate, given the availability of programmable DNA binding proteins such as the nuclease-dead version of CRISPR Cas9 (dCas9) . A model system was designed to explore this idea in which dCas9 from Streptococcus pyogenes was fused to the C-terminus of e-NpuN cage while the NpuC cage construct was fused to the N-terminus of histone H3 within a nucleosome (Figure a, Figure S3).…”