Expression and purification of recombinant human CUL2 from Escherichia coli cells

Abstract

Cullin-2 (CUL2) based cullin RING ligases (CRL2) comprise a family of ubiquitin E3 ligases that exist only in multi-cellular organisms and are crucial for cellular processes such as embryogenesis and viral pathogenesis. In the CRL2 complex, CUL2 serves as a docking platform for the substrate receptor module, which consists of the adaptor proteins Elongin B and Elongin C (EloB/C) as well as the tumor suppressor protein VHL, where VHL is known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Abnormalities in HIF1α degradation can result in vascular tumor cell proliferation and cause a rare disease known as von-Hippel-Lindau (VHL) syndrome. Because of its critical role in HIF1α ubiquitination, CRL2VHL has been investigated for the development of novel therapeutics and treatments against disease. Given the importance of CRL2s in biological and biomedical research, a method that efficiently produces functional recombinant human CUL2 proteins will greatly facilitate studies on CRL2-dependent protein ubiquitination and degradation. Previous studies have generated recombinant human CUL2 using a baculovirus expression system. However, this method is very costly and time consuming for optimal protein expression in insect cells. To achieve a more cost-effective way of producing the human CUL2 protein, we propose to generate recombinant CUL2 in E. coli cells. We suggest that removal of unstructured regions in CUL2 would increase its solubility in E. coli. Additionally, we explored and applied the “Split-n-Co-express” strategy as previously used for CUL1 and CUL3. We report that each of our strategies resulted in soluble CUL2 expressed in E. coli cells, and following a series of purification by chromatography, the purity of the recombinant protein reached ~95%. Using in vitro assays, we found that our purified CUL2 could conjugate to NEDD8, a key activator of CRLs, and that the neddylated CUL2 could bind to VHL•EloB/C to ubiquitinate the HIF1α degron peptide. The binding and enzymatic activity of our recombinant protein demonstrate that we have established a new system for the expression and purification of functional human CUL2 from E. coli cells.