Abstract
Cullin-2 (CUL2) based cullin-RING ligases (CRL2s) 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. CUL2 is the scaffold protein that binds one of the interchangeable substrate receptor modules, which consists of adaptor proteins and the substrate receptor protein. The VHL protein is a substrate receptor known to target hypoxia-inducible factor α (HIF1α) for ubiquitination and degradation. Because of its critical role in the ubiquitination of important cellular factors such as HIF1α, CRL2s have been investigated for their biological functions and the development of novel therapeutics against diseases. Given the importance of CRL2s in biological and biomedical research, methods that efficiently produce functional CUL2 proteins will greatly facilitate studies on the mechanism and regulation of CRL2s. Here, we report two cost-effective systems for the expression and purification of recombinant human CUL2 from E. coli cells. The purified CUL2 proteins were ~ 95% pure, could bind their substrate receptor modules, and were enzymatically active in transferring ubiquitin or ubiquitin-like protein to the corresponding substrate in in vitro assays. The presented methodological advancements will help advance research in CRL2 function and regulation.
Highlights
Protein turnover is a cellular regulatory system defined by the continuous synthesis and decomposition of specific proteins to maintain the integrity of optimally functioning p roteins[1,2]
Co-opting cells’ proteolytic machineries for therapeutic benefit, re-directing E3 ligases towards new substrates using PROteolysis TArgeting Chimeras (PROTACs), is emerging as a promising pharmacological s trategy11–14. CRL2VHL has been on the forefront of protein-degrader drug development, with numerous von Hippel-Lindau (VHL)-based PROTACs discovered in the past decade[15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]
The low protein solubility is a common limitation for high-level expression of full-length cullin proteins in bacteria[35], and this limitation was overcome by deleting two short unstructured regions when full-length human CUL1RBX1 was produced in E. coli 39
Summary
Protein turnover is a cellular regulatory system defined by the continuous synthesis and decomposition of specific proteins to maintain the integrity of optimally functioning p roteins[1,2]. Abnormalities during protein turnover, during protein degradation, often result in human diseases such as cystic fibrosis and liposarcoma. Cullin-2 (CUL2) is one type of cullin protein, which only exists in multi-cellular o rganisms[4,8]. It plays critical roles in both physiological processes such as embryogenesis, and pathogenesis of the human immunodeficiency virus (HIV-1)[4]. Because CRL2s play critical roles in protein ubiquitination and drug discovery, mechanistic insights into the activity and regulation of CRL2 will benefit the development of novel therapeutics against disease and infection. We have established and optimized an efficient system for bacterial expression and purification of functional recombinant human CUL2RBX1 proteins
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