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

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 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 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 towards 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.