Target Validation and E3 Ligase Selection

VHL and CRBN are the substrate receptors of two multi-subunit cullin-RING E3 ubiquitin ligases, CRL2VHL and CRL4^CRBN, which direct substrate-specific ubiquitination and proteasomal degradation. At the start of the development of a degrader, two points should be confirmed: whether the target protein can be ubiquitinated by a system that can be reconstituted in vitro, and which E3 ligase and matching E2 partner will carry the reaction. Most published degraders recruit CRBN or VHL, while programs built on other ligases establish the cullin scaffold, the adaptor subunits and the matching E2 partner at the enzyme level. Amerigo Scientific supplies these components with defined composition, including assembled CRL2^VHL and CRL4^CRBN complexes, TR-FRET assays for E1 activation and E1-to-E2 transfer, and profiling panels covering 24 E2 conjugating enzymes.

Recombinant E3 Ligase Complexes

CRBN and VHL are substrate receptors that assemble into cullin-RING ligases. Ubiquitin transfer occurs when the RING subunit positions a charged E2 next to the recruited substrate, so catalytic readouts require the cullin scaffold and RBX1, while binding and structural readouts can be run with the receptor module alone.

We provide assembled complexes with defined subunit stoichiometry to support ubiquitin transfer in a reconstituted reaction as well as binding measurements.

Applications

• Protein degradation
• PROTAC and Molecular Glue discovery
• Selectivity profiling

E1 and E1/E2 Activity Assays

Ubiquitin reaches a substrate through a fixed relay. The E1 enzyme charges ubiquitin, transfers it to an E2 conjugating enzyme, and the E3 ligase then directs the charged E2 to the substrate. Activity at these first two steps underlies every downstream ubiquitination experiment.

We provide plate-based TR-FRET kits for the two upstream steps. Both use terbium as the donor fluorophore, whose long-lived fluorescence allows the FRET signal to be read after a time delay, which reduces the contribution from buffers, proteins and chemical compounds present in the sample. The assay can be run in a 96-well or 384-well format and produces a reproducible signal with a Z' > 0.7.

E2-E3 Pairing Profiling

Roughly forty E2 conjugating enzymes serve several hundred E3 ligases, and each ligase works with a restricted subset of them. The E2 also shapes chain architecture, and since K48-linked chains route substrates to the proteasome while K63-linked chains carry signaling roles, E2 identity governs both the rate of ubiquitination and its biological consequence.

Kits that assay 24 E2 enzymes in parallel against a ligase of interest under identical reaction conditions, are available in chemiluminescent and colorimetric formats. Colorimetric format is suitable for laboratories equipped with a standard absorbance plate reader. Colorimetric readouts are more consistent day-to-day than luminescent-based detection methods, fast, and easy to use.

Features

• Testing functionality of over 24 E2's with an E3 ligase of interest
• Ready-to-assay. Contains 24 human E2's pre-plated in triplicate with E1 and Ubiquitin.
• Detection system provides robust readout for E3 ligase activity.
• The kit utilizes non-radioactive reporter substrates.
• Uses native, rather than tagged, ubiquitin

E2 Enzymes in the Plate

Custom E2 Enzyme Panels

Once profiling has narrowed the field, routine assays need the productive E2 enzymes in working quantities. Consistent preparation across those enzymes keeps later comparisons internally controlled.

A panel of four E2 enzymes selected by the user and prepared under a common process. It supplies either the shortlist that emerges from a profiling experiment or a hypothesis-driven set chosen from published ligase and E2 relationships.

Applications

• Identification of cognate E2 enzymes for E3 ligases
• Demonstration of novel E2 or E3 activity with cognate enzyme
• High-throughput screening of agonists/antagonists of either E2 or E3 activity
• Determination of Michaelis constant (Km) for E2:E3 interactions