Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.
Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin.
复制标题
泛素位点特异性突变体的基因合成、表达、结构和功能活性。
DOI：
10.1016/s0021-9258(18)47925-2
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
L. Mueller
中科院分区：
文献类型：
--
作者：
D. Ecker;T. Butt;J. Marsh;E. Sternberg;N. Margolis;B. Monia;S. Jonnalagadda;M. Khan;P. L. Weber;L. Mueller
为了研究泛素的结构和功能，我们化学合成了一个泛素基因，该基因编码动物泛素的氨基酸序列，插入一系列限制性内切酶位点，将该基因分成八个“诱变模块”。构建了一系列位点特异性突变来选择性地干扰分子的各个区域。突变基因在大肠杆菌中大量表达，并纯化了修饰蛋白。为了确定氨基酸取代对结构的影响，利用二维核磁共振研究了泛素的溶液结构，并对每个突变蛋白进行了结构扰动筛选。除了一个例外，除了突变点之外，几乎没有发现任何变化。利用泛素激活酶E1和网状细胞蛋白降解实验对突变蛋白进行功能研究，以确定泛素在细胞内蛋白水解中活性的重要分子区域。
To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight “mutagenesis modules.” A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.