Nature Chemical Biology, 29 August, 2025, DOI：https://doi.org/10.1038/s41589-025-02019-7
ALFA nanobody-guided endogenous labeling
Zhe Wang, Fang Hu, Fudong Xue, Wenting He, Lin Yuan & Pingyong Xu
Abstract
Small peptide tags offer advantages as their compact size reduces target protein interference, making them valuable for labeling endogenous proteins. However, the lack of inherent fluorescence poses challenges for post-genome knockin monoclonal clone screening. Here we report an adaptable approach leveraging antigen-stabilizing fluorescent protein-fused nanobodies (Nbs) to selectively illuminate cells with successful ALFA tag knockins, streamlining high-throughput cell screening using fluorescence-activated cell sorting. Through targeted mutations and screening of ALFA Nbs (NbALFA), the fluorescently labeled Nb can be selectively degraded in the absence of the ALFA peptide. Conversely, successful insertion of the ALFA peptide into the genome results in a substantial increase in the fluorescence intensity of the Nb. This technique, termed ALFA Nb-guided endogenous labeling (ANGEL), enables a wide array of versatile applications within the native cellular environment. These applications include precise protein labeling and signal amplification through the tandem arrangement of ALFA tags, dynamic monitoring of protein behavior, initiation of protein degradation processes and analysis of protein interactome.
文章链接：https://www.nature.com/articles/s41589-025-02019-7
相关报道：//m.wyreworks.com/jz/zxdt/202509/t20250904_7959270.html
附件下载：