Epitope-specific enzymes are powerful tools for site-specific protein modification but generally require genetic manipulation of the target protein. Here, we describe the laboratory evolution of the bacterial transpeptidase sortase A to recognize the LMVGG sequence in endogenous amyloid-β (Aβ) protein. Using a yeast display selection for covalent bond formation, we evolved a sortase variant that prefers LMVGG substrates from a starting enzyme that prefers LPESG substrates, resulting in a >1,400-fold change in substrate preference. We used this evolved sortase to label endogenous Aβ in human cerebrospinal fluid, enabling the detection of Aβ with sensitivities rivaling those of commercial assays. The evolved sortase can conjugate a hydrophilic peptide to Aβ₄₂, greatly impeding the ability of the resulting protein to aggregate into higher-order structures. These results demonstrate laboratory evolution of epitope-specific enzymes toward endogenous targets as a strategy for site-specific protein modification without target gene manipulation and enable potential future applications of sortase-mediated labeling of Aβ peptides.

表位特异性酶是位点特异性蛋白质修饰的强大工具，但通常需要对目标蛋白质进行遗传操作。在这里，我们描述了细菌转肽酶分选酶 A 的实验室进化，以识别内源性淀粉样蛋白-β (Aβ) 蛋白中的 LMVGG 序列。使用酵母展示选择共价键形成，我们从偏好 LPESG 底物的起始酶进化出偏好 LMVGG 底物的分选酶变体，导致底物偏好发生 >1,400 倍的变化。我们使用这种进化的分选酶来标记人脑脊液中的内源性 Aβ，从而能够以与商业化验相媲美的灵敏度检测 Aβ。进化的分选酶可以将亲水肽与 Aβ _42结合，极大地阻碍了所得蛋白质聚集成高级结构的能力。这些结果证明了表位特异性酶向内源性靶标的实验室进化，作为一种无需靶基因操作的位点特异性蛋白质修饰策略，并使分选酶介导的 Aβ 肽标记成为可能的未来应用。