Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies
Chemical ligation of folded recombinant proteins: Segmental isotopic labeling of domains for NMR studies
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DOI：
10.1073/pnas.96.2.388
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发表时间：
1999-01-19
影响因子：
11.1
通讯作者：
Muir, TW
中科院分区：
文献类型：
--
作者：
Xu, R;Ayers, B;Muir, TW
已经开发了一种方便的体外化学连接策略，其允许折叠的重组蛋白连接在一起。该策略允许产物的片段性、选择性同位素标记。Abelson蛋白酪氨酸激酶的src同源性3型和2型结构域（SH 3和SH 2）构成了蛋白的调节装置，它们分别以反应形式制备，可以在正常的蛋白折叠条件下连接在一起，在连接点形成正常的肽键。该策略用于制备Abelson蛋白酪氨酸激酶-SH（32）结构域对的NMR样品量，其中仅一个结构域用N-15标记，质谱和NMR分析用于确认连接的蛋白质的结构，其也显示出适当的配体结合性质。通过使用融合蛋白的表达和体外化学连接的组合来制备具有选择性标记的具有单位点突变的区段的重组蛋白的能力将增加用NMR方法在溶液中进行蛋白质结构测定的尺寸限制。表达的蛋白质结构域的体外化学连接也将提供合成连接的蛋白质结构域的组合方法。
A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together, This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with N-15, Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.