Program in Cellular and Molecular Medicine , Boston Children's Hospital, Harvard Medical School , Boston , Massachussets 02115 , United States.
Institute for Molecular Bioscience , The University of Queensland , Brisbane , QLD 4072 , Australia.
J Am Chem Soc. 2019 Oct 30;141(43):17388-17393. doi: 10.1021/jacs.9b09166. Epub 2019 Oct 16.
Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant asparaginyl endopeptidase, AEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by AEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.
具有特定底物特异性的蛋白质连接酶是蛋白质工程的通用工具。在反应完成后，目前报道的蛋白质连接酶的产物包含被同一连接酶识别的氨基酸序列，导致连接和水解作为竞争反应的反复循环。因此，以前在不同位置顺序标记蛋白质的努力需要具有正交特异性的连接酶。一种重组天冬酰胺内肽酶 AEP1 对进入的亲核试剂具有混杂性。这种混杂性使我们能够定义一种由天然氨基酸组成的亲核试剂，该亲核试剂有效地连接到底物上，但产生的产物被 AEP1 识别不良。用有效识别模块修饰的蛋白质可以很容易地进行修饰，以得到带有切割抗性基序的定义产物，而含有这种较差识别基序的蛋白质则基本保持不变。我们展示了这种方法可获得的 N-或 C-末端蛋白质修饰的多功能性，并以优异的收率以顺序、位点特异性的方式对单个底物蛋白的 N-和 C-末端进行修饰。
