Recombinant CRBNmidi (S41-E187_GSG_D249-I426), Cereblon, E3 Ligase

CRBNmidi (Kroupova et al., 2024) is a truncated CRBN construct that enables sufficient expression of soluble and stable protein in E. coli without additional factors. The truncated CRBNmidi still retains functionality similar to full-length wild-type CRBN and is therefore ideal for crystallographic and biophysical studies of CRBN-based PROTACs.

CRBNmidi contains the Lon protease-like domain (Lon), spanning residues 41 - 187, followed by a Gly-Ser-Gly (GSG) linker that partially replaces the HB domain and connects the Thalidomide Binding Domain (TBD), which spans residues 249-426. The CRBNmidi construct also includes 12 stabilizing mutations (C78I, I92V, K116N, Q134E, R283W, C287N, V293S, G302D, L342R, C343E, T359I, L423I).

Protein Construct: S_A41-E187/D249-I426
Source: Human
Expression Host: Escherichia coli
Molecular weight [kDa]: 37.44
pI: 5.8
Extinction Coefficient [M^-1cm^-1]: 46410
Concentration: 3.6 mg/mL
Storage Buffer: 20 mM HEPES pH 7.5, 500 mM NaCl, 0.5 mM TCEP
Tag: none

Amino acid sequence:

QC Data: Datasheet
Purity SDS: >95 %
Hydrodynamic Radius (nm): 3.34
Polydispersity Index (PDI): 0.24
T_on (°C): 40.5
T_m (°C): 41.49
Calculated Mass [Da]: 34325.368
Measured Mass [Da]: 34323.39

Structure and function

Cereblon (CRBN) functions as the substrate receptor of the Cullin 4–RING E3 ubiquitin ligase complex by coupling target recognition to ubiquitin transfer. Through its C-terminal thalidomide-binding domain, CRBN directly engages its substrates, bringing them and ubiquitin-loaded E2 into close proximity and positioning the target for polyubiquitination.

Its native targets include many important endogenous substrates, such as adenosine monophosphate-activated protein kinase subunit α1, amyloid precursor protein or glutamine synthetase.

CRBN as important drug target

CRBN and its associated E3 ubiquitin ligase are central to targeted protein degradation strategies with significant therapeutic potential. Immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide act as molecular glues that enable CRBN to recruit new substrates (neosubstrates), leading to their ubiquitination and degradation, which has diverse anti-cancer and immunmodulatory effects.

Moreover, CRBN-binding ligands have been adapted for the development of PROTACs that induce the degradation of specific clinically relevant targets, underscoring the importance of CRBN in modern drug discovery.