Open AccessSortase-Mediated Phage Decoration for Analytical Applications
Author(s) -
Ding Yuan,
He Chen,
Jiao Li,
Lianrun Huang,
Guangyue Song,
Zhenfeng Li,
Xiude Hua,
Gualberto GonzálezSapienza,
Bruce D. Hammock,
Minghua Wang
Publication year - 2021
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.1c02322
Subject(s) - phage display , chemistry , sortase a , peptide , immunoassay , conjugate , peptide library , rhodamine , phagemid , combinatorial chemistry , detection limit , bacteriophage , microbiology and biotechnology , antibody , chromatography , biochemistry , peptide sequence , fluorescence , escherichia coli , bacterial protein , mathematical analysis , physics , mathematics , quantum mechanics , biology , immunology , gene
Phage-borne peptides and antibody fragments isolated from phage display libraries have proven to be versatile and valuable reagents for immunoassay development. Due to the lack of convenient and mild-condition methods for the labeling of the phage particles, isolated peptide/protein affinity ligands are commonly removed from the viral particles and conjugated to protein tracers or nanoparticles for analytical use. This abolishes the advantage of isolating ready-to-use affinity binders and creates the risk of affecting the polypeptide activity. To circumvent this problem, we optimized the phage display system to produce phage particles that express the affinity binder on pIII and a polyglycine short peptide fused to pVIII that allows the covalent attachment of tracer molecules employing sortase A. Using a llama heavy chain only variable domain (VHH) against the herbicide 2,4-D on pIII as the model, we showed that the phage can be extensively decorated with a rhodamine-LPETGG peptide conjugate or the protein nanoluciferase (Nluc) equipped with a C-terminal LPETGG peptide. The maximum labeling amounts of rhodamine-LPETGG and Nluc-LPETGG were 1238 ± 63 and 102 ± 16 per phage, respectively. The Nluc-labeled dual display phage was employed to develop a phage bioluminescent immunoassay (P-BLEIA) for the detection of 2,4-D. The limit of detection and 50% inhibition concentration of P-BLEIA were 0.491 and 2.15 ng mL -1 , respectively, which represent 16-fold and 8-fold improvement compared to the phage enzyme-linked immunosorbent assay. In addition, the P-BLEIA showed good accuracy for the detection of 2,4-D in spiked samples.