Prokaryotic Expression, Purification, Crystallization Of Yeast Doa1(Ufd3) Protein And Analysis Of Doa1 Structure

Protein degradation mediated by ubiquitin-proteasome pathway is an important mechanism which modulates cellular protein activity and function. Doal, belonging to PLAA family proteins, was first linked to the ubiquitin proteasome pathway.It is known to play a key role during ubiquitin-dependent protein degradation, and can bind directly to ubiquitin and Cdc48 to facilitate Cdc48 recruitment of ubiquitin.Cdc48 is an AAA-ATPase molecular chaperone that can interact with ubiquitin-binding cofactors to facilitate its cellular functions.In this paper,we will solve the structure of yeast Doal through structure biology methods to clarify the functional mechanism of Doal which participate in Cdc48-mediated ubiquitination pathway and DNA damage pathway.In this paper,in order to solve the structure of Doal(Ufd3)(320-C),We cloned the gene of yeast Doal(Ufd3)(320-C) into PET28a plasimid and then 6HIS-tagged Doal(320-C)was expressed in E.coli. Recombinant protein was first purified on a Ni²⁺ affinity column.Further purification of Doal(Ufd3)(320-C) was carried out by Ion Change Chromatography and Molecular Exclusion Chromatography.Crystals were grown by the hanging drop vapor diffusion method. The crystal structure of Doal(Ufd3)(320-C) was determined by SAD method.The results of this research include:1.Gene cloning of yeast Doal(Ufd3)(320-C) into PET28a and pGEX-6P-l.2.Recombinant Doal(Ufd3)(320-C) protein purification to high purity.3.Well-diffracted crystals growing and data collection.4. Structure determination of Doal(Ufd3)(320-C) at the first time around the world.5.Structural analysis and residues that may involve in binding to Cdc48determiantion by in vitro GST pull-down experiments.These results are considerably useful for the furher study of functional mechanism of Doal(Ufd3) in Cdc48-mediated ubiquitination pathway.