Scalable insect cell expression and purification screening applied to CRL4-DCAF substrate receptors

The ubiquitin-proteasome system is one of the primary mechanisms responsible for degradation of intracellular proteins. Cullin-RING E3 ligases (CRL) are modular, multi-subunit complexes that catalyse ubiquitination of a wide variety of proteins, marking them for degradation by the proteasome. Substrate specificity is conferred by the substrate receptor subunit of the CRL, of which there are hundreds. Targeted protein degradation (TPD) is a drug modality that involves hijacking the activity of CRLs to ubiquitinate non-native neosubstrates via compound-induced ternary complex formation between a substrate receptor and the target. Of the many CRL substrate receptors, the DDB1- and Cul4-associated factor (DCAF) family are of high interest and potential for TPD. To enable characterisation of DCAF proteins and ligand screening campaigns, we have undertaken high-throughput recombinant protein expression screening in insect cells and small-scale plate-based purification of 24 DCAF proteins to identify soluble recombinant protein. Co-expression with the stabilising substrate adaptor DDB1 is required for, or enhances, expression of many DCAFs and provides a folding quality control measure through co-purification with tagged DCAF protein. Of 13 DCAF proteins that had not previously been expressed in the literature, we identify 8 that express well as promising candidates for scale-up. We provide sequence and construct information as a resource for the community. This screening method could be expanded to more DCAF proteins and applied to other CRL substrate receptor families. • Expression and purification screening enables soluble expression of DDB1- and Cul4-associated factor (DCAF) proteins • 54 constructs across 24 DCAF proteins were screened using a plate-based workflow • Co-expression and co-purification with DDB1 can enhance expression and report on DCAF folding • Eight previously unexpressed DCAFs show promise for scale-up and further study • Recombinant DCAF expression resources support characterisation and ligand discovery for targeted protein degradation