Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d -Aminopeptidase
Quantitative N- or C-Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d -Aminopeptidase
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使用分选酶和 d-氨基肽酶对带有未激活肽的蛋白质进行定量 N 或 C 末端标记
DOI：
10.1002/ange.202310862
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发表时间：
2024
影响因子：
--
通讯作者：
Arnott Z
中科院分区：
文献类型：
--
作者：
Arnott Z
蛋白质的定量和选择性标记广泛用于学术和工业实验室，并且使用转肽酶（例如分选酶）催化标记蛋白质已被证明是用于这种选择性修饰的流行策略。这类酶的一个主要挑战是，大多数程序需要过量的标记试剂或活化底物，而不是简单的商业来源的肽。我们报告了偶联酶策略的使用，该策略能够使用未活化的标记肽定量标记蛋白质的N-和C-末端。氨肽酶与转肽酶联合使用允许肽副产物的序列特异性降解，使平衡移动以有利于产物形成，这大大提高了反应效率。随后的反应优化允许使用基本上等摩尔比的肽标记物与蛋白质的N-末端标记蛋白质，以及仅少量过量的C-末端标记。最大限度地减少定量标记所需的底物量有可能改善工业过程，并促进转肽作用作为蛋白质标记方法的使用。
Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases, such as sortases, has proved to be a popular strategy for such selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using unactivated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibrium to favor product formation, which greatly enhances the reaction efficiency. Subsequent optimisation of the reaction allows N‐terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.