Devany Matthew, Kotharu N Prasad, Matsuo Hiroshi
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455, USA.
Protein Expr Purif. 2005 Apr;40(2):244-7. doi: 10.1016/j.pep.2004.07.008.
The 375 amino acid human protein DEK has been expressed in two functional, structured domains. DEK is an abundant nuclear protein that associates with chromatin and alters its topology by introducing positive supercoiling in DNA, which results in lower replication efficiency. DEK has clinical importance as transfection of the cDNA of the C-terminal region of DEK can partially reverse the abnormal DNA-mutagen sensitivity in fibroblasts derived from ataxia-telangiectasia (A-T) patients, and elevated levels of DEK mRNA are observed in various forms of cancer. Because high-level expression of full-length DEK has proved elusive, we sought an alternative for structural studies that would provide insights on DEK's function. We have discovered that DEK contains two structured domains and have expressed these domains at a high level in Escherichia coli in M9 minimal media. The N-terminal domain (amino acids 68-226) includes the region responsible for introducing supercoils into DNA, and the C-terminal domain (amino acids 309-375) includes the region that can reverse the abnormal DNA-mutagen sensitivity of A-T cells. 1H-15N correlation nuclear magnetic resonance spectra of these two fragments reveal the characteristic signature of folded proteins.
由375个氨基酸组成的人类蛋白质DEK已在两个功能性的结构化结构域中表达。DEK是一种丰富的核蛋白，它与染色质结合，并通过在DNA中引入正超螺旋来改变其拓扑结构，从而导致较低的复制效率。DEK具有临床重要性，因为转染DEK C端区域的cDNA可部分逆转共济失调毛细血管扩张症（A-T）患者成纤维细胞中异常的DNA诱变敏感性，并且在各种癌症中都观察到DEK mRNA水平升高。由于全长DEK的高水平表达难以实现，我们寻求一种用于结构研究的替代方法，以深入了解DEK的功能。我们发现DEK包含两个结构化结构域，并已在M9基本培养基中在大肠杆菌中高水平表达了这些结构域。N端结构域（第68至226位氨基酸）包括负责将超螺旋引入DNA的区域，C端结构域（第309至375位氨基酸）包括可以逆转A-T细胞异常DNA诱变敏感性的区域。这两个片段的1H-15N相关核磁共振谱揭示了折叠蛋白的特征信号。
