CUEDC2的表达纯化与溶液结构初步探索

发布时间：2018-08-11 20:09

【摘要】： CUEDC2 (CUE Domain Containing 2)是一个功能未知的蛋白质。最近研究发现CUEDC2能够和孕激素受体结合,促进孕激素诱导的受体的泛素化和降解,可能作为一个新的肿瘤治疗靶点,因而具有十分重要的应用价值。为了更好的研究其生物学功能,我们拟用核磁共振方法研究其三维结构。通过生物信息学的分析表明CUEDC2包含一个CUE结构域。CUE结构域是一个约40个氨基酸的广泛存在于真核蛋白质中的结构域。CUE结构域具有多种功能,其中包括内质网上所合成的折叠错误和序列错误的蛋白质的降解过程。CUE结构域有着与UBA结构域相似的结构,都有三个α-螺旋折叠而成。CUE结构域的α-螺旋1中的保守的MFP motif和α-螺旋3中的LL motif与泛素的疏水表面相互作用,同时据文献报道CUE结构域可以形成二聚体,可以附加的结合多个泛素,因而具有较高的泛素亲和能力。许多包含CUE结构域的蛋白质既能识别单泛素化又能识别多泛素化。实验证明,许多包含CUE结构域的蛋白质都能够以一种CUE结构域依赖性的方法发生泛素化。CUEDC2是一个分子量约为32 kDa包含287个氨基酸的酸性蛋白质。由于分子量较大,我们计划将其截成2个片段,分别解析其溶液结构。本文以CUEDC2为研究对象,分别构建了多个CUEDC2的截断体,并通过优化各种表达与纯化条件,获得了高表达的,且符合核磁共振实验要求的蛋白质样品溶液,并利用多维核磁共振技术对CUEDC2 N端包含133个氨基酸残基的结构域的溶液结构进行了初步的研究与探索。首先,我们表达、纯化了GST-CUEDC(1-133),但是在用凝血酶切除GST标签蛋白时,我们发现在用凝血酶酶切16小时后仍然无法将GST标签蛋白切下。于是我们考虑构建有较小的标签的克隆。我们将CUEDC2 N端的133个氨基酸构建到pET-28a载体上,并对其表达、纯化条件及蛋白质序列进行优化。最终获得了折叠状态较好的CUEDC(1-133)cut蛋白质样品。之后,我们优化了核磁共振的实验温度并确定了三共振实验条件。我们采集了一系列的三维核磁共振实验图谱,包括用于主链谱峰指认的HNCA、HNCOCA、CBCANH、CBCA(CO)NH和用于侧链谱峰指认的HBHA(CO)NH、CCCONH、15N-NOESY-HSQC、HCCH-COSY和HCCH-TOCSY谱。然后我们利用NMRPipe软件包对谱图进行处理,利用CARA软件归属了大部分主链原子的化学位移。本文还对CUEDC2(133-287)蛋白进行了初步的研究。表达、纯化了GST-CUEDC(133-287)。并利用凝血酶对GST-CUEDC(133-287)酶切,除去了GST标签蛋白,获得了高浓度的CUEDC(133-287)蛋白样品。然后我们1H核磁共振谱对其结构进行初步的评价。结果表明可能由于CUEDC2(133-287)这个截断体中缺失了某个关键的氨基酸序列,导致其折叠构象改变,蛋白质折叠不完全。我们希望通过构建其它的CUEDC2截断体来得到C末端折叠较好的CUEDC2蛋白,进行结构研究。
[Abstract]:CUEDC2 (CUE Domain Containing 2) is an unknown protein. Recently, it has been found that CUEDC2 can bind to progesterone receptor and promote the ubiquification and degradation of progesterone-induced receptors, which may be a new target of tumor therapy, so it has very important application value. In order to better study its biological function, we intend to use nuclear magnetic resonance method to study its three-dimensional structure. Bioinformatics analysis shows that CUEDC2 contains a CUE domain. Cue domain is a domain of about 40 amino acids, which widely exists in eukaryotic proteins. This includes the degradation of proteins synthesized by the endoplasmic reticulum with folding errors and sequence errors. The cue domain has a structure similar to that of the UBA domain. The conserved MFP motif in 伪 -helix 1 and LL motif in 伪 -helix 3 interact with the hydrophobic surface of ubiquitin. It is reported that the CUE domain can form dimers. It can attach multiple ubiquitin, so it has higher Ubiquitin affinity. Many proteins containing CUE domains can recognize both monoubiquitization and polyubiquitin. It has been proved that many proteins containing CUE domain can produce ubiquification in a CUE domain dependent way. CUEDC2 is an acidic protein with a molecular weight of 32 kDa containing 287-amino acids. Due to their high molecular weight, we plan to cut them into two fragments and analyze their solution structures respectively. In this paper, the truncation of CUEDC2 was constructed, and the high expression protein sample solution was obtained by optimizing the expression and purification conditions. The solution structure of 133-amino acid residues at the N-terminal of CUEDC2 was studied by using multi-dimensional NMR technique. First, we expressed and purified GST-CUEDC (1-133), but when we removed the GST tagged protein with thrombin, we found that the GST tag protein could not be removed after 16 hours of thrombin digestion. So we consider building clones with smaller tags. We constructed 133 amino acids from the N-terminal of CUEDC2 into pET-28a vector and optimized their expression, purification conditions and protein sequence. Finally, CUEDC (1-133) cut protein samples with good folding state were obtained. After that, we optimized the experimental temperature of NMR and determined the experimental conditions of triple resonance. We have collected a series of 3D NMR spectra, including HNCAA HNCOCACCAN CBCACA (CO) NH used for the identification of the main chain peaks and HBHA (CO) NH + CCCONHN 15N-NOESY-HSQCU HCCH-COSY and HCCH-TOCSY spectra used to identify the side chain peaks of HCCH-COSY. Then we use the NMRPipe software package to process the spectra and use the CARA software to assign the chemical shifts of most of the main chain atoms. CUEDC2 (133-287) protein was also studied. Expression and purification of GST-CUEDC (133-287). GST-CUEDC (133-287) was digested with thrombin to remove the GST labeled protein, and a high concentration of CUEDC (133-287) protein was obtained. Then our 1H NMR spectra were used to evaluate its structure. The results suggest that the folding conformation of CUEDC2 (133-287) is not complete due to the deletion of a key amino acid sequence in the truncation. We hope to construct other CUEDC2 truncates to obtain a better C-terminal folded CUEDC2 protein and study its structure.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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