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Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

ABSTRACT: Quenchbody (Q-body) is a quench-based fluorescent immunosensor labeled with fluorescent dye(s) near the antigen-binding site of an antibody. Q-bodies can detect a range of target molecules rapidly and directly. However, because Q-bodies show different antigen responses depending on the antibody used, time-consuming optimization of the Q-body structure is often necessary, and a high-throughput screening method for discriminating and selecting good Q-bodies is required. Here, we aimed to develop a molecular display method of nanobody-based “mini Q-bodies” by combining yeast surface display and coiled-coil forming E4/K4 peptide-based fluorescence labeling. As a result, the yeast-displayed mini Q-body recognizing the anti-cancer agent methotrexate (MTX) showed significant quenching and MTX-dependent dequenching on cells. To demonstrate the applicability of the developed method to select highly responsive mini Q-bodies, a small nanobody library consisting of 30 variants that recognize human serum albumin was used as a model. The best variant, showing a 2.4-fold signal increase, was obtained through selection by flow cytometry. Furthermore, the same nanobody prepared from Escherichia coli also worked as a mini Q-body after dye labeling. The described approach will be applied to quickly obtain well-behaved Q-bodies and other fluorescent biosensors for various targets through directed evolutionary approaches.

SUBMITTER: Inoue A

PROVIDER: S-EPMC8604967 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Phosphatidylinositides are important signaling molecules that interact with a myriad of cellular proteins, many of which remain unidentified. We previously screened a yeast surface displayed human proteome library to identify protein fragments with affinity for the phosphatidylinositides, phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate. Much of the diversity in the screened selection outputs was represented by clones present at low frequencies, suggesting that a significant number of additional phosphatidylinositide-binding protein fragments might be present in the selection outputs. In the studies described in this report, we developed a novel cDNA library analysis method and comprehensively analyzed the polyclonal selection outputs from the phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3,4,5-trisphosphate selections using a high-density exon microarray. In addition to the nine previously reported phosphatidylinositide-binding protein fragments, we identified 37 new phosphatidylinositide-binding candidates. Nine of 37 contain known phosphatidylinositide-binding domains, whereas the remaining 28 contain no known phosphatidylinositide-binding domain. We cloned and confirmed phosphatidylinositide binding by fluorescence-activated cell sorting for 17 of these novel candidate protein fragments. Our experiments suggest that phosphatidylinositide binding by these 17 novel protein fragments is dependent on both the inositol phosphate "headgroup" and the lipid "tail." This is in contrast with the PH domain containing fragments we tested, for which the inositol phosphate headgroup was sufficient for binding. The novel PtdIns-binding fragments come from a wide variety of proteins, including splicing factors, transcription factors, a kinase, and a polymerase. Intriguingly, 11 of the phosphatidylinositide-binding protein fragments are from nuclear proteins, including four containing homeobox domains. We found that phosphatidylinositides and double-stranded DNA oligonucleotides derived from homeobox domain target sequences compete for binding to homeobox domain-containing protein fragments, suggesting a possible mechanism for phospholipid-dependent transcriptional regulation. FACS enrichment of target-binding clones in yeast human cDNA display libraries coupled with comprehensive analysis of the selection output by DNA microarray analysis is an effective method for investigating common as well as rare protein interactions. In particular, this method is well suited for the study of small molecule/protein and drug/protein interactions.

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Evaluation and selection of potent fluorescent immunosensors by combining fluorescent peptide and nanobodies displayed on yeast surface

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