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PreprintCited by 0Viral Infectious Diseases and Gene Expression in InsectsUbiquitin and proteasome pathwaysProtein Degradation and Inhibitors

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

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Cowan, Angus D., Jaekel, Stefan, Ciulli, Alessio·2025·Protein Expression and Purification
DOI 10.64898/2025.12.04.690205

Preprint — published as 10.1016/j.pep.2026.106907

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Abstract

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.

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Metadata

Authors
Cowan, A. D., Jaekel, S., & Ciulli, A.
Affiliations
University of Dundee (GB)
Corresponding author
  • Cowan, Angus D.
  • Ciulli, Alessio
ORCID
Venue
Protein Expression and Purification
Publisher
openRxiv
Published
2025-12-05
Volume
241
Pages
106907-106907
ISSN
1046-5928 (ISSN-L)
DOI
10.64898/2025.12.04.690205
OpenAlex
W7133512048
Cited by
0
References
1
FWCI
0.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Protein Expression and Purification (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
Funders
  • Innovative Medicines Initiative 2 (IMI2) Joint Undertaking (875510)
  • Wellcome Trust (100476/Z/12/Z, 094090/Z/10/Z, 097945/C/11/Z)
  • European Commission (101024945)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Degradation and Inhibitors
Keywords
Cullin-RING E3 ligases, targeted protein degradation, DDB1, insect cell expression, protein solubility, protein folding quality control, ligand screening, ubiquitin-proteasome system
MeSH terms
Animals, Carrier Proteins, DNA-Binding Proteins, Humans, Recombinant Fusion Proteins, Recombinant Proteins, Substrate Specificity, Spodoptera, Ubiquitin-Protein Ligases, Sf9 Cells

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