BioInvenu’s proprietary LinkLight™ technology offers innovative tools for cell-based protein-protein interaction assays.
The LinkLight™ assay consists of two components. A protein A is linked to a Tobacco Etch Virus (TEV) protease and a protein B is linked to a permuted luciferase (pLuc). An inactive pLuc has been created by breaking luciferase into two fragments, rearranging the fragment order in that the N-terminal fragment is moved to C-terminus and C-terminal fragment is moved to the N-terminus and reconnecting them by a TEV protease cleavage sequence. Upon interaction between protein A and B, inactive pLuc is cleaved, the cleaved luciferase fragments are spontaneously refolded, driven by fragment self-complementation affinity, and active luciferase is reconstituted. The signal is specific and sensitive for protein A and B interaction. Other reporter proteins can be permuted to replace the pLuc. The LinkLight assay is not another enzyme or protein fragment complementation (EFC or PFC) methods. LinkLight technology overcomes the significant drawback of enzyme or protein fragment spontaneous self-complementation. The spontaneous fragment self-complementation caused by the high affinity of two fragments increases false interactions and background signals, and makes transient protein-protein interactions as constitutive interactions. Furthermore, LinkLight assay technology has a unique ability in that one protein tagged with TEV can interact with multiple proteins tagged with permuted reporters to perform multiplex assays.
LinkLight™ technology provides simple, robust, economic, and sensitive methods to detect molecules modulating protein-protein interactions in live cells with immediate and stable signals. LinkLight™ technology does not involve transcription or translation signal cascades, therefore false and off-target signals are greatly reduced. The technology is also different from protein-fragment complementation assays (PCA) or enzyme-fragment complementation assays (ECA) in which fragment irreversible self-complementation could produce high background signals, especially when proteins are over-expressed or two fragments have high complementation affinity. LinkLight™ assays can generate stable signals even from rapid transient protein-protein interactions. LinkLight assay also avoids drawbacks of BRET assay regarding spectrum separation, spatial distance and orientation requirements between a bioluminescent donor and a fluorescent acceptor, light intensity, donor/acceptor ratio, and unable for establishing stable cell line assays. In addition, the detection substrate, luciferin, is more economical and simple to use. Furthermore, the technology does not need expensive equipment, as LinkLight™ assays can be read on many luminescence readers.
LinkLight™ technology utilizes protein-protein interactions as functional signal readouts. The assay system can theoretically be used to find molecules that modulate protein-protein interaction or directly block protein-protein interactions. The technology can be applied to a broad range of protein-protein interactions by engineering appropriate fusion protein partners, such as cell membrane and cytoplasmic protein interactions, membrane, and membrane protein interactions, cytoplasmic protein interactions, nuclear protein interactions, and cell-cell interaction through membrane protein interactions.
Reporter proteins that generate a fluorescent, colorimetric, luminescent, or cell survival readout can be permuted to substitute the permuted luciferase in the LinkLight™ technology. Currently, firefly luciferase, renilla luciferase, and beta-lactamse have been utilized as the reporters in the LinkLight™assay systems. BioInvenu will expand the LinkLight™ assay technology and offers comprehensive assay products and services.
Advantages of LinkLight™ Assay Technology
- Specific: only TEV tagged receptors give signals, endogenous receptors or receptor family members do not interfere.
- Sensitive: Bioluminescent signals are more sensitive than other methods.
- Short exposure times: no transcription & translation of a reporter gene, less off-target effects.
- Economic: detection reagent (luciferin) is commercial available, the operation cost is a fraction of competitor’s prices.
- Immediate & physiological relevant signals: the cell-based assays generate stable and immediate signals for either transient or stable protein-protein interactions.
- Robotic adaptability: the assays are very simple to operate and ready for high through-put screenings.
- A Cell-based Protein-Protein Interaction Method Using A Permuted Luciferase
- ERK and β-arrestin interaction: a converging-point of signaling pathways for multiple types of cell-surface receptors (J Biomol Screen 1087057114557233, first published on October 31, 2014)
- Poster: GPCR LinkLight Assay (SLAS 2012)