Prokaryotic Expression, Purification, Crystallization Of Dcn1p In Protein Neddylation And Analysis Of The Proteins Interacted

Protein degradation mediated by ubiquitin-proteasome pathway is an important mechanism which modulates cellular proteins' activity and function. Modification of Cullins by Nedd8 is important for their ubiquitin E3 ligase activities. Dcnlp, an evolutionarily conserved protein, promotes Cullin neddylation in vivo, binds directly to Nedd8 and associates with Cullinl in the budding yeast Sacchromyces cerevisiae.In this paper, in order to know more about the functions of Dcnlp, first recombinant full-length yeast Dcnlp as a GST-fusion protein. GST-Dcnlp was expressed in E.coli. Recombinant was purified on a glutathione-agarose (GSH) affinity column, followed by removal of the GST-tag with thrombin digestion. Further purification of Dcnlp was carried out using successive chromatography steps on Q and Superdex-200 columns. Crystals were grown by the hanging drop vapor diffusion method. GST-pull down technology and western blotting was used to screen the proteins interacted with Dcnlp. Finally, used surface plasmon resonance technology (SPR) to validate this interaction and get the kinetic constants of the interaction in vitro.The results of this research include:1. Get Dcnlp protein with the concentration above 95%;2. Best diffracting crystals were obtained in a condition containing 100mM Tris HCl (Ph8.5), 30% PEGMME-5000 and 0.2 M ammonium sulfate.3. Solved the structure of The Dcnlp at the first time around the world. 1.9 A-resolution structure of yeast Dcnlp shows that the region encompassing residues 66-269 adopts a rectangular parallelepiped-like overall fold, consisting of elevenα-helix motifs.4. First put forward that Dcnlp interacts with Rbx-lp directly in vitro, and obtained the kinetic constants of the interaction KA=3.72×10~5. This observation is consistent with and extends a previous observation that Dcnlp, Cdc53p and Rbx-lp can coexist in a multiprotein complex.These results together suggest that DCN-1 functions as a component of the Nedd8 E3 ligase complex, which is considerably useful for the further studying of ubiquitin-proteasome pathway.