BUILD YOUR OWN ASSAY TOOLS

Enzyme Fragment Complementation (EFC) Technology Assay Principle

The EFC technology is based on two recombinant β-galactosidase (β-gal) fragments – a large protein fragment (enzyme acceptor, EA) and a small peptide fragment (enzyme donor, ED), which are added as tags onto proteins in your assay or during the detection step. Separately, the β-gal fragments are inactive, but when combined as part of the assay application (e.g. ligand induced protein-protein interaction), they form an active β-gal enzyme. The enzyme can then hydrolyze a substrate (provided within the detection reagents) to produce a chemiluminescent signal that can be read on any standard luminometer. This simplistic system allows for interrogation of biomolecular interactions for characterizing targets and pathways as well as advancing therapeutic screening and development programs.

When creating your own EFC cell-based assay, you will need the following tools:

• Cell culture and transfection reagents including tissue culture plates, media, and a facility appointed with proper tissue culture equipment and instrumentation (e.g. a standard luminometer).
• A plasmid to introduce your target of interest to be tagged with ED. Your target could be a GPCR, kinase, ion channel, transporter, nuclear protein, or other protein of interest
• Optional: A parental cell line containing a reporter protein tagged with EA. The reporter protein is related to the type of assay being performed. For example, when looking at internalization of a membrane receptor protein to the endosome, the reporter protein would be an endosomal protein. Alternatively, for a GPCR β-arrestin recruitment assay your reporter protein would be β-arrestin.
• EFC detection reagents that includes the enzyme substrate for producing the chemiluminescent signal.

Typical workflow protocol schematic to show how to generate an EFC cell-based assay starting with a parental cell line. Start with an engineered PathHunter® parental cell line containing EA to study subcellular translocation (e.g. including membrane receptor trafficking), SH2 recruitment for kinases or cytokine receptors, or GPCR β-arrestin recruitment. The specific parental cell line chosen determines the assay type. Then, create an ED-tagged target protein plasmid followed by transfection of this plasmid into the parental cell line containing EA. Perform antibiotic selection, detect the level of expression of the two fusion proteins (optional), and perform the desired assay in the presence of a ligand.

Create Your Own Trafficking Assays Using Engineered Parental Cell Lines

Generate a PathHunter trafficking assay by first creating a plasmid vector with your target protein of choice tagged with the Enzyme Fragment Complementation (EFC) β-galactosidase (β-gal) enzyme donor (ED). Simply transfect a PathHunter EA parental cell line with the plasmid and perform a trafficking assay in the presence of a ligand. In this example, the Endosome-EA (ENDO-EA) PathHunter EA parental cell line is used, and the target protein is a GPCR. The assay detects the trafficking of the GPCR from the plasma membrane to the endosome upon ligand (pharmacochaperone) binding to GPCR. Once the GPCR moves to the endosome, the ED-tagged GPCR fuses with the large EFC enzyme acceptor (EA) that is bound to an endosomal reporter protein. Upon addition of the β-gal substrate and formation of the active enzyme (ED+EA), the enzyme hydrolyzes its substrate to produce a chemiluminescence signal. For creating a trafficking assay from the endoplasmic reticulum (ER) to the plasma membrane, use the Membrane-EA (MEM-EA) PathHunter EA parental cell line (see an example of this in the following customer publication).

Create Your Own GPCR β-Arrestin Cell-Based Assays in Any Dividing Cell Type

Create your own GPCR β-arrestin cell-based assays to evaluate ligand-induced β-arrestin recruitment to any GPCR in any dividing cell type. Using the Enzyme Fragment Complementation technology, simply infect your target cells with the PathHunter β-Arrestin Retroparticles, transfect the cells with a GPCR plasmid, and perform a PathHunter GPCR β-arrestin assay with your ligand of interest. Note: β-Arrestin2-EA Retroparticles and GPCR-PK can be introduced into the target cells in either order.

Create Your Own Cell-Based Cytotoxicity Assays in Any Dividing Cell Type

Target cells expressing the receptor antigen of choice can be engineered to stably express a protein (housekeeping protein) tagged with the small enzyme donor (called the enhanced ProLabel (ePL) or β-gal reporter fragment) using KILR Retroparticles. When the stable target cell line is used in an EFC-based cytotoxicity assay (e.g., ADCP, ADCC, CDC), the EFC reaction can be measured with the addition of the large enzyme acceptor (EA) β-gal fragment that creates an active β-gal enzyme. With the addition of hydrolyzing substrate, a chemiluminescent output can be detected on any bench top luminometer.

Create Your Own SH2-protein Recruitment Assays Using Engineered Parental Cell Lines

Generate your own PathHunter SH2-protein recruitment assay using the enzyme fragment complementation (EFC) technology. Create a plasmid vector with your target protein of choice (e.g. RTK in this example) tagged with the small enzyme donor (ED), ProLink^™ , of the EFC β-galactosidase (β-gal). Simply transfect this plasmid into a PathHunter SH2-recuitment parental cell line containing an SH2 protein tagged with the large enzyme acceptor (EA) and perform a kinase functional or activity assay in the presence of a ligand.