Split‐BioID: a proximity biotinylation assay for dimerization‐dependent protein interactions

Munter, Sofie De; Görnemann, Janina; Derua, Rita; Lesage, Bart; Qian, Junbin; Heroes, Ewald; Waelkens, Etienne; Eynde, Aleyde Van; Beullens, Monique; Bollen, Mathieu

doi:10.1002/1873-3468.12548

Cited by 90 publications

(71 citation statements)

References 25 publications

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“…Alternatively, tagging through genome editing would ensure physiological expression levels. Of note, as we observed stronger re-activation with our split-BioID assay based on the E256/G257 splitting site than with the alternative E140/Q141 splitting site12, it will most probably be better suited when low expression levels of the fusion proteins are desired.…”

Section: Discussionmentioning

confidence: 76%

“…This lower activity was sufficient for performing BioID experiments (see below). A recent study also described a PCA based on two BirA* fragments using an alternative splitting site (E140/Q141)12. We wondered if this splitting site would perform similar to our PCA.…”

Section: Resultsmentioning

confidence: 87%

“…Indeed, by selectively activating Ago with either Dicer or TNRC6 using split-BioID, we could specifically analyse the RLC or the miRISC and identify a previously unknown regulator of miRNA-mediated translational repression. Another study recently described an alternative split-BioID assay12, suggesting that BirA* can be split at multiple sites.…”

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confidence: 99%

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Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

et al. 2017

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Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein–protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.

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Section: Discussionmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 87%

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Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

et al. 2017

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“…Novel or improved applications using BioID ligase have spurred numerous follow-up articles including a smaller version of BioID with improved sensitivity and localization (22), its use for identifying protein-RNA interactions (23), split-BioID studies (24,25), and faster versions of BioID (26). Several conventional approaches have been utilized to stably introduce BioID-fusion proteins to cells including transfection (7), viral infection (27), and more recently, CRISPR-Cas9 (28).…”

Section: Introductionmentioning

confidence: 99%

BioID as a Tool for Protein-Proximity Labeling in Living Cells

Sears

May

Roux

2019

Methods in Molecular Biology

107

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BioID has become an increasingly utilized tool for identifying candidate protein-protein interactions (PPIs) in living cells. This method utilizes a promiscuous biotin ligase, called BioID, fused to a protein-of-interest that when expressed in cells can be induced to biotinylate interacting and proximate proteins over a period of hours, thus generating a history of protein associations. These biotinylated proteins are subsequently purified and identified via mass spectrometry. Compared to other conventional methods typically used to screen strong PPIs, BioID allows for the detection of weak and transient interactions within a relevant biological setting over a defined period of time. Here we briefly review the scientific progress enabled by the BioID technology, detail an updated protocol for applying the method to proteins in living cells, and offer insights for troubleshooting commonly encountered setbacks.

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“…This proximal biotinylation has been successfully used to identify novel protein interactions [see reviews: 4, 9], and has been shown as complementary to traditional affinity purification approaches [10]. Interests in this approach have led to improved variants of BioID proteins: BioID2, a smaller biotin ligase from Aquifex aeolicus [11], and a split-BioID as a dimerization dependent biotin ligase [12, 13]. …”

Section: Introductionmentioning

confidence: 99%

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat

Cheah

Yamada

2017

Biochemical and Biophysical Research Communications

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Protein-protein interactions are the molecular basis of cell signaling. Recently, proximity based biotin identification (BioID) has emerged as an alternative approach to traditional co-immunoprecipitation. In this protocol, a mutant biotin ligase promiscuously labels proximal binding partners with biotin, and resulting biotinylated proteins are purified using streptavidin conjugated beads. This approach does not require preservation of protein complexes in vitro, making it an ideal approach to identify transient or weak protein complexes. However, due to the high affinity bond between streptavidin and biotin, elution of biotinylated proteins from streptavidin conjugated beads requires harsh denaturing conditions, which are often incompatible with downstream processing. To effectively release biotinylated proteins bound to streptavidin conjugated beads, we designed a series of experiments to determine optimal binding and elution conditions. Interestingly, the concentrations of SDS and IGEPAL-CA630 during the incubation with streptavidin conjugated beads were the key to effective elution of biotinylated proteins using excess biotin and heating. This protocol provides an alternative method to isolate biotinylated proteins from streptavidin conjugated beads that is suitable for further downstream analysis.

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Split‐BioID: a proximity biotinylation assay for dimerization‐dependent protein interactions

Cited by 90 publications

References 25 publications

Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

BioID as a Tool for Protein-Proximity Labeling in Living Cells

A simple elution strategy for biotinylated proteins bound to streptavidin conjugated beads using excess biotin and heat

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