Expression And Purification Of CUEDC2 And The Preliminary Investigation Of Its Solution Structure

CUE Domain Containing 2 (CUEDC2 ) is a new protein with unidentified function. Recently, CUEDC2 was found to interact with progesterone receptor (PR) and promotes progesterone-induced PR degradation by the ubiquitin-proteasome pathway. CUEDC2 may be a new target for cancer therapeutics. The solvation of the solution structure of CUEDC2 will facilitate the research of its function.Based on bioinformatic analysis, CUEDC2 was considered as a CUE domain-containing protein. The CUE domain is moderately conserved consisting of 40 amino acids and is structurally related to the ubiquitin-binding UBA domain, which is found in a variety of eukaryotic proteins.The CUE domain has been found in proteins with a variety of functions, including the degradation of misfolded proteins in the endoplasmic reticulum and protein sorting. The CUE domain is folded into a three-helix bundle. A conserved MFP motif inα-helix1 and an LL motif inα-helix 3 interact with the conserved hydrophobic patch of ubiquitin. It has been reported that the CUE domain exists as a domain-swapped dimmer to make additional contacts with ubiquitin, resulting in higher binding affinity with ubiquitin. Proteins containing CUE domains, such as Vps9 and Cue1, can bind ubiqutin in mono and polyubiquitin manners. Some CUE-containing proteins are ubiquitinylated in a CUE domain-dependent manner. CUEDC2 is an acidic protein consisting of 287 amino acids with a molecular weight of 32 kDa. The full length protein is too large for NMR study and was thus divided into separate N-terminal domain (1-133) and C-terminal domain (133-287) for solution structure determination.In this thesis have been described the structural studies of CUEDC2.A few truncated forms of CUEDC2 were constructed, expressed in E. coli and purified with optimized conditions. High quality protein samples suitable for NMR experiments were obtained. Preliminary results of the solution structure of the N-terminal domain CUEDC2(1-133) have been obtained based on heteronuclear multidimensional NMR experiments.First of all, the CUEDC2(1-133) was expressed as GST-fusion protein. But it turned out to be difficult to cut off GST protein tag with thrombin even after 16 h incubation. Therefore, a new construct of CUEDC2(1-133) was made from pET-28a plasmid, resulting in a His-tag fusion protein of CUEDC2(1-133). The conditions for the expression and purification of this fusion protein were optimized. Finally, a protein sample of CUEDC2(1-133) was obtained with good folding as checked by NMR experiments. The NMR experimental conditions were optimized and a set of 3D NMR experiments were carried out, which including HNCA, HN(CO)CA, CBCANH, CBCA(CO)NH for the assignments of backbone, and HBHA(CO)NH, CC(CO)NH, 15N-NOESY-HSQC, HCCH-COSY, HCCH-TOCSY for theassignments of sidechain atoms. All the spectra were processed with NMRPipe Software Package, and chemical shift assignments were carried out using CARA and NMRView. Most of the backbone signals have been assigned.The C-terminal domain GST-CUEDC2(133-287) has also been expressed as GST fusion protein and purified with GST-tag removed. with the preliminary 1H NMR spectra showed that CUEDC2(133-287) was not well folded. It might be due to the lack of key amino acids in this truncated form. New constructs of this truncated domain have been carrying on to solve this problem.