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.
Highlights
Description of a set of lentiviral constructs for doxycycline-inducible BioID. All vector configurations enable amino- or carboxyl-terminal fusion in a Gateway system
Generation of Lentiviral Vector Sequences and Expression Constructs—The region internal to the long terminal repeats (LTRs) of the second generation pLVX-Tight-Puro lentiviral backbone (Takara Bio, The abbreviations used are: affinity purification (AP)-Mass spectrometry (MS), affinity-purification coupled to mass spectrometry; BirA*, E. coli biotin ligase harboring a R118G mutation; VP16, herpes simplex virus (HSV) virion protein 16; rTetR, reverse tetracycline repressor; rtTA, reverse tetracycline-controlled transactivator, rTetR fused to VP16; PGK, phosphoglycerate kinase 1 promoter; short variant of the elongation factor-1 alpha (sEF1a), short form of elongation factor-1 alpha (EF1a), promoter; Mouse Embryonic Fibroblasts (MEF), mouse embryonic fibroblast; MCS, multiple cloning site; NLS, nuclear localization sequence
Consistent with these functions, BioID of H2B led to the identification of a set of proteins enriched for the Gene Ontology (GO) CC term “Chromosome” (GO: 0005694; p value Յ 6.9 ϫ 10Ϫ8 across all cell lines) and the GO Biological Process (BP) term “Chromosome Organization” (GO:0006325; p value Յ 2.0 ϫ 10Ϫ15 across all cell lines; supplemental Table S6)
Summary
The proximity-dependent biotinylation approach BioID is increasingly used to define proximity interactomes and subcellular organization, but is most often limited to a few transfectable 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 cloned a cassette consisting of BirA* (enabling BioID) and a single FLAG epitope (that can be used both to detect bait expression and to perform AP-MS) [31] downstream of a tetracycline-regulatable promoter, enabling inducible gene expression We inserted this cassette into five lentiviral vector backbones, each harboring different features, including: integrated rtTA (tetracycline-controled transactivator), puromycin selection or mCitrine expression. We outline strategies for lentiviral backbone selection and considerations for experimental design that will assist the community in the use of these reagents
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