A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria Journal Article


Authors: Fay, A.; Kurland, A. P.; Li, Z.; Monetti, M.; Johnson, J. R.; Glickman, M. S.
Article Title: A split ALFA tag-nanobody system for protein localization and proximity proteomics in mycobacteria
Abstract: Tuberculosis remains a globally significant infection, and new insights into the biology of Mycobacterium tuberculosis are badly needed. Discovery of protein localization and protein complex composition are powerful approaches to determine protein function but have not been widely applied in mycobacteria, in part due to technical barriers. Here we develop a multifunctional system that utilizes the ALFA tag and functional protein fusions to an anti-ALFA nanobody (NBALFA) to target proteins in fast- and slow-growing mycobacteria. Insertion of the ALFA epitope tag on the target protein, coupled with conditional expression of the NBALFA fused to a fluorescent protein, faithfully recapitulates cytosolic and membrane protein localization by fluorescent microscopy in living cells. Targeted NBALFA can relocalize an ALFA-tagged protein to inclusion bodies or the cytoplasmic membrane, demonstrating enforced protein localization. Finally, the conditional expression of the NBALFA fused to TurboID for proximity proteomics allowed the identification of known partner proteins of the RNA polymerase complex and the PKS13 mycolic acid biosynthesis protein. We conclude that the split ALFA tag-nanobody system is a flexible platform for discovering protein biology in mycobacteria. IMPORTANCE: This study establishes a new platform for discovery proteomics in mycobacteria using a new nanobody-based approach. The findings will be of interest for all bacteriologists as the approach will be applicable to a variety of microbial systems. This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine
Keywords: genetics; metabolism; proteomics; bacterial protein; chemistry; mycobacterium tuberculosis; bacterial proteins; recombinant fusion proteins; protein transport; mycobacterium; fusion protein; procedures; protein-protein interactions; nanobody; single-domain antibodies
Journal Title: mBio
Volume: 16
Issue: 8
ISSN: 2150-7511
Publisher: American Society for Microbiology  
Date Published: 2025-08-01
Start Page: e0097125
Language: English
DOI: 10.1128/mbio.00971-25
PUBMED: 40576343
PROVIDER: scopus
PMCID: PMC12345155
DOI/URL:
Notes: The MSK Cancer Center Support Grant (P30 CA008748) is acknowledge in the PDF -- Corresponding authors is MSK author: Michael S. Glickman -- Source: Scopus
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MSK Authors
  1. Allison J Fay
    16 Fay
  2. Michael Glickman
    114 Glickman
  3. Zhuoning Li
    21 Li