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Laboratory evolution of a sortase enzyme that modifies amyloid-β protein.

修饰淀粉样蛋白-β 蛋白的分选酶的实验室进化。

  • 影响因子:9.33
  • DOI:10.1038/s41589-020-00706-1
  • 作者列表:"Podracky CJ","An C","DeSousa A","Dorr BM","Walsh DM","Liu DR
  • 发表时间:2021-03-01
Abstract

: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.

摘要

: 表位特异性酶是用于位点特异性蛋白质修饰的强大工具,但通常需要靶蛋白的遗传操作。在这里,我们描述了细菌转肽酶sortase A识别内源性淀粉样蛋白-β (A β) 蛋白中LMVGG序列的实验室进化。使用共价键形成的酵母展示选择,我们从优选LPESG底物的起始酶中进化出优选LMVGG底物的分选酶变体,导致底物偏好的> 1,400倍变化。我们使用这种进化的分选酶来标记人脑脊液中的内源性a β,使a β 的检测能够与商业测定的灵敏度相媲美。进化的分选酶可以将亲水性肽与Aβ42缀合,极大地阻碍了所得蛋白质聚集成更高级结构的能力。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,作为无需靶基因操作的位点特异性蛋白质修饰的策略,并使分选酶介导的a β 肽标记的潜在未来应用成为可能。

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