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

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• The ubiquitin-proteasome system degrades intracellular proteins.
• Cullin-RING E3 ligases (CRLs) are crucial for protein ubiquitination and degradation.
• DDB1- and Cul4-associated factors (DCAF) are key substrate receptors for CRLs and hold potential for targeted protein degradation (TPD) drug development.

Purpose of the Study:

• To enable characterization of DCAF proteins for TPD.
• To screen for soluble recombinant DCAF proteins for ligand screening campaigns.

Main Methods:

• High-throughput screening of recombinant protein expression in insect cells.
• Small-scale, plate-based purification of 24 DCAF proteins.
• Co-expression with DDB1 to enhance DCAF solubility and stability.

Main Results:

• Identified 8 novel, soluble DCAF proteins from 13 previously unexpressed candidates.
• Demonstrated that co-expression with DDB1 is essential for or enhances the expression of many DCAFs.
• Established a scalable method for DCAF protein expression and purification.

Conclusions:

• The identified DCAF proteins are promising candidates for TPD drug development.
• The developed screening method is effective for identifying soluble DCAF proteins.
• This approach can be expanded to other CRL substrate receptor families and applied to broader TPD research.