Sachdeva Amit, Wang Kaihang, Elliott Thomas, Chin Jason W
Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
J Am Chem Soc. 2014 Jun 4;136(22):7785-8. doi: 10.1021/ja4129789. Epub 2014 May 23.
Rapid, one-pot, concerted, site-specific labeling of proteins at genetically encoded unnatural amino acids with distinct small molecules at physiological pH, temperature, and pressure is an important challenge. Current approaches require sequential labeling, low pH, and typically days to reach completion, limiting their utility. We report the efficient, genetically encoded incorporation of alkyne- and cyclopropene-containing amino acids at distinct sites in a protein using an optimized orthogonal translation system in E. coli. and quantitative, site-specific, one-pot, concerted protein labeling with fluorophores bearing azide and tetrazine groups, respectively. Protein double labeling in aqueous buffer at physiological pH, temperature, and pressure is quantitative in 30 min.
在生理pH值、温度和压力条件下，利用不同的小分子对基因编码的非天然氨基酸处的蛋白质进行快速、一锅法、协同、位点特异性标记是一项重大挑战。目前的方法需要顺序标记、低pH值，而且通常需要数天才能完成，这限制了它们的实用性。我们报告了使用优化的大肠杆菌正交翻译系统，在蛋白质的不同位点高效地、基因编码地掺入含炔烃和环丙烯的氨基酸，以及分别用带有叠氮化物和四嗪基团的荧光团进行定量、位点特异性、一锅法、协同蛋白质标记。在生理pH值、温度和压力条件下，在水性缓冲液中进行蛋白质双标记，30分钟内即可完成定量。
