A Versatile Lentiviral Delivery Toolkit for Proximity-dependent Biotinylation in Diverse Cell Types

Mol Cell Proteomics. 2018 Nov;17(11):2256-2269. doi: 10.1074/mcp.TIR118.000902. Epub 2018 Jul 10.

Abstract

Proximity-dependent biotinylation strategies have emerged as powerful tools to characterize the subcellular context of proteins in living cells. The popular BioID approach employs an abortive E. coli biotin ligase mutant (R118G; denoted as BirA*), which when fused to a bait protein enables the covalent biotinylation of endogenous proximal polypeptides. This approach has been mainly applied to the study of protein proximity in immortalized mammalian cell lines. To expand the application space of BioID, here we describe a set of lentiviral vectors that enable the inducible expression of BirA*-tagged bait fusion proteins for performing proximity-dependent biotinylation in diverse experimental systems. We benchmark this highly adaptable toolkit across immortalized and primary cell systems, demonstrating the ease, versatility and robustness of the system. We also provide guidelines to perform BioID using these reagents.

Keywords: Affinity proteomics; Affinity tagging; Cell biology; Cellular organelles; Molecular biology; Protein-Protein Interactions; Spectral counting.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotinylation
  • Gene Transfer Techniques*
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Lentivirus / metabolism*
  • Male
  • Mice
  • Neoplasm Proteins / metabolism
  • Promoter Regions, Genetic / genetics
  • Reproducibility of Results
  • Transgenes

Substances

  • Neoplasm Proteins