Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins
Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins
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开发用于酶促合成氟化酪氨酸及其掺入蛋白质的单一培养物大肠杆菌表达系统
DOI：
10.1016/j.jfluchem.2022.110014
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发表时间：
2022
影响因子：
1.9
通讯作者：
Pomerantz, William C.K.
中科院分区：
文献类型：
--
作者：
Olson, Noelle M.;Johnson, Jorden A.;Peterson, Kerstin E.;Heinsch, Stephen C.;Marshall, Andrew P.;Smanski, Michael J.;Carlson, Erin E.;Pomerantz, William C.K.
目前的实验依赖于生物合成代谢蛋白质标记与19 F通常需要氟化氨基酸，这在2-和3-氟酪氨酸的情况下可能是昂贵的。然而，使用这些氨基酸为蛋白质动力学，结构和功能提供了有价值的见解。在这里，我们开发了一种新的细胞内方法，用于从容易获得的取代酚生成氟化酪氨酸，并随后在单一细菌表达培养物中对蛋白质进行代谢标记。该方法使用编码模型蛋白BRD 4（D1）和来自弗氏柠檬酸杆菌的酪氨酸苯酚裂解酶的双基因质粒，所述酪氨酸苯酚裂解酶催化从苯酚、丙酮酸和铵形成酪氨酸。通过19 F NMR和LC-MS方法分析，我们的系统证明了酶促氟酪氨酸的产生和19 F标记蛋白的表达。我们的系统的进一步优化应该提供一个具有成本效益的替代各种传统的蛋白质标记策略。
Current experiments that rely on biosynthetic metabolic protein labeling with19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase fromCitrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of19F-labeled proteins as analyzed by19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.