Transpeptidation-Mediated Assembly of Tripartite Split\nGreen Fluorescent Protein for Label-Free Assay of Sortase Activity

Abstract

Transpeptidation\nof surface proteins catalyzed by the transpeptidase\nsortase plays a critical role in the infection process of Gram-positive\npathogen, and probing sortase activity and screening its inhibitors\nare of great significance to fundamental biological research and pharmaceutical\ndevelopment, especially novel antivirulence drug design. Herein, we\ndeveloped a novel fluorescent biosensor to detect sortase activity\nbased on a transpeptidation-triggered assembly of tripartite split\ngreen fluorescent protein (split GFP). Peptide P₁, composed\nthe 10th β-sheet of GFP (GFP10) and the sortase A (SrtA) recognition\nsequence (LPETX), and peptide P₂, the 11th β-sheet\nof GFP (GFP11) with oligoglycine at N-terminal, were designed and\nsynthesized, respectively. Existence of SrtA enables P₁ and P₂ to ligate into one peptide, which could spontaneously\nbind to GFP1-9 (the 1st to 9th β-sheets of GFP) and assemble\ninto functional GFP. Thus, the sortase-catalyzed transpeptidation\ncan switch on the fluorescence signal of GFP. The method was successfully\napplied to detect SrtA activity with a low detection limit of 0.16\nnM and for its inhibition measurement. Moreover, the feasibility of\nthe proposed assay was further expanded to detect SrtA in human blood\nand further Gram-positive pathogens analysis in frozen food. Our method,\nusing tripartite split GFP as a readout, is facile, label-free, and\nsensitive and exhibits great potential as a promising platform for\nsortase detection and inhibitor screening.