Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation
Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation
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DOI：
10.1021/ja203111c
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发表时间：
2011-08-03
影响因子：
15
通讯作者：
Chin, Jason W.
中科院分区：
文献类型：
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作者：
Nguyen, Duy P.;Elliott, Thomas;Chin, Jason W.
我们报告了进化的正交吡咯赖氨酰-tRNA合成酶/tRNA(CUA)对，其指导N(ε)-L-硫杂丙酰-L-赖氨酸、N(ε)-D-半胱氨酰-L-赖氨酸和N(ε)-L-半胱氨酰-L-赖氨酸高效、位点特异性地掺入大肠杆菌中的重组蛋白中。我们证明，通过我们的方法引入的独特的 1,2-氨基硫醇可以通过氰基苯并噻唑缩合有效、快速、特异性地标记，从而将生物物理探针定量引入蛋白质中。此外，我们表明，与半胱氨酸标记相结合，这种方法允许在两个不同的、遗传定义的位点用不同的探针对蛋白质进行双重标记。
We report evolved orthogonal pyrrolysyl-tRNA synthetase/tRNA(CUA) pairs that direct the efficient, site-specific incorporation of N(epsilon)-L-thiaprolyl-L-lysine, N(epsilon)-D-cysteinyl-L-lysine, and N(epsilon)-L-cysteinyl-L-lysine into recombinant proteins in Escherichia coli. We demonstrate that the unique 1,2-aminothiol introduced by our approach can be efficiently, rapidly, and specifically labeled via a cyanobenzothiazole condensation to quantitatively introduce biophysical probes into proteins. Moreover, we show that, in combination with cysteine labeling, this approach allows the dual labeling of proteins with distinct probes at two distinct, genetically defined sites.