Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584CG, Utrecht, The Netherlands.
Angew Chem Int Ed Engl. 2020 Dec 1;59(49):21870-21874. doi: 10.1002/anie.202003779. Epub 2020 Sep 28.
Genetic code reprogramming is a powerful approach to controlled protein modification. A remaining challenge, however, is the generation of vacant codons. We targeted the initiation machinery of E. coli, showing that restriction of the formyl donor or inhibition of the formyl transferase during in vitro translation is sufficient to prevent formylation of the acylated initiating tRNA and thereby create a vacant initiation codon that can be reprogrammed by exogenously charged tRNA. Our approach conveniently generates peptides and proteins tagged N-terminally with non-canonical functional groups at up to 99 % reprogramming efficiency, in combination with decoding the AUG elongation codons either with native methionine or with further reprogramming with azide- and alkyne-containing cognates. We further show macrocyclization and intermolecular modifications with these click handles, thus emphasizing the applicability of our method to current challenges in peptide and protein chemistry.
遗传密码重编程是一种控制蛋白质修饰的强大方法。然而，仍然存在一个挑战，那就是产生空位密码子。我们针对大肠杆菌的起始机制进行了研究，结果表明，在体外翻译过程中限制甲酰供体或抑制甲酰转移酶足以防止酰化起始 tRNA 的甲酰化，从而产生空位起始密码子，该密码子可被外源充电的 tRNA 重新编程。我们的方法方便地生成肽和蛋白质，在高达 99%的重编程效率下，在 N 端标记非经典功能基团，同时解码 AUG 延伸密码子，要么使用天然蛋氨酸，要么使用含有叠氮化物和炔烃的类似物进一步重编程。我们还展示了这些点击手柄的大环化和分子间修饰，从而强调了我们的方法在肽和蛋白质化学当前挑战中的适用性。
