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Expression and purification of functional recombinant Cullin1-Rbx1/2 in E. coli
Fangyu Zhao1, Chuntong Li2, Xu Li3
1Beijing Life Science Academy, Beijing, 102209, China; Center for BioAnalytical Chemistry, Hefei National Laboratory of Physical Science at Microscale, University of Science and Technology of China, Hefei, 230026, China; Institute of Biomedical Engineering, College of Life Sciences, Qingdao University, Qingdao, 266071, China.
None:
Protein ubiquitination is a crucial post-translational modification in eukaryotes that is mediated by E1-E2-E3 enzymatic cascades and regulates a wide range of cellular processes. Cullin-RING E3 ligases (CRLs) represent the largest family of RING-type E3 ligases and play pivotal roles in these processes. Generating full-length, active Cullin1-Rbx1 (CRL1) is essential for biochemical and biophysical investigations. In this study, we developed an efficient strategy to produce functional CRL1 complexes from E. coli, by fusing an MsyB protein to N-terminus of Cullin1 to improve its solubility. The recombinant CRL1 demonstrated full functionality, successfully assembling with substrate receptor Skp1-Skp2-Cks1 and mediating the polyubiquitination of Ub-p27-degron substrate. Using recombinant CRL1, we found that phosphorylation at Ser65 inhibited the CRL1-UBE2R1 mediated ubiquitin chain elongation on Ub-p27-degron. Furthermore, using the same strategy, we successfully generated Cullin1-Rbx1 mutants and Cullin1-Rbx2 complexes, thereby expanding the applicability of our method. Collectively, this work establishes a rapid and cost-effective platform for the production of CRL1 complexes, facilitating structural and functional studies.
