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Development and performance of BibID method

Part 4. Development of a GFP nanobody-directed Proximity Biotinylation Assay based on the Bimolecular Fluorescence Biotinylation Assay based on the Bimolecular Fluorescence

4.3.2 Development and performance of BibID method

Given the target protein-specific PL of TurboID, we conceived a BiFC-based TurboID system, BibID, which enabled the binary protein complex-specific PL in living cells. This method takes advantage of both GFP-nanobody and TurboID, schematically represented in Figure II-8. The nanobody was first reported in 1993, asa specific class of light chain-deleted antibodies in camels (Hamers-Casterman et al., 1993). A nanobody is strictly monomeric, highly stable, and generally smaller, thus it is far more efficient than classical antibodies. Besides, as GFP is the most important genetic marker for biological research, several different GFP nanobodies (GBP) have been developed for targeting and binding GFP or its variants (Fridy et al., 2014; Kubala et al., 2010; Twair et al., 2014). More recently, conditionally stable GFP-binding nanobody (csGBP) was described, which enabled detection of less noisy GFP-tagged proteins, notably, along with their analysis showing that unbound csGBP was efficiently degraded by the proteasome (Ariotti et al., 2018). By combining the csGBP with TurboID, the BibID system facilitates an improved signal-to-noise (S/N) ratio for a specific PL detection. Furthermore, csGBP straddles the split site in commonly used BiFC pairs (Kubala et al., 2010), such as the one recruited in our system, CC155/VN173. The folding induced by resulting BiFC is absolutely required for csGBP binding, leading that recognition of the unfolded halves by csGBP will not occur theoretically.

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Figure II-8. Schematic representation of BibID system. The binding between csGBP and folded CC/VN stabilises TurboID-csGBP, which leads to proximity biotinylation around the CC/VN-tagged POIs, here referred as A/B protein complex. The unbound TurboID-csGBP will be degraded by the ubiquitin proteasome system, which would largely alleviate non-specific labelling when csGBP-CC/VN binding saturates. The biotin labelled proteins can be subsequently isolated by biotin affinity purification and identified by LC-MS/MS analysis. HA, HA epitope tag. csGBP, conditionally stable GFP-binding nanobody. CC, C-terminal fragment of mCerulean (155-238aa). VN, N-terminal fragment of mVenus (1-172aa).

As proof-of-concept, N-terminal split mVenues (VN173) and C-terminal split mCerulean were used to construct BiFC system with one well-studied PPI, HOXA9/PBX1, generating VN-HOXA9 and CC-PBX1. Meanwhile, the modular TurboID-csGBP fusion was made and tagged by HA-tag. To validate the system efficiency, a preliminary immunofluorescence test was carried out in HEK cells by transfection comparing with several control settings (Figure II-9). The EGFP was coexpressed with TurboID-csGBP as system positive control. VN-HOXA9 and CC-PBX1 were exchanged in reciprocal experiment, cotransfected with TurboID-csGBP, as two negative controls of csGBP. One

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system negative control was also designed to monitor the background of PL, using only TurboID-csGBP alone transfection. The complete BibID system utilized a 3-plasmid transfection, similar to previous single TurboID test, showing a highly biotin-dependent PL profile (Strep-A555 channel, Figure II-9B). The resulting PL was observed for tight association of VN-HOXA9/CC-PBX1 interaction or EGFP expression, as opposed to just one-half of the interaction pair, such as VN-HOXA9 or CC-PBX1 only (Strep-A555 vs.

BiFC/EGFP, Figure II-9B). Interestingly, in the absence of folded CC/VN or EGFP, the TurboID-scGBP expression was still generally visible in the control conditions regardless of biotin addition. These background-like weak HA signals are likely caused by TurboID-csGBP en route to proteasomal degradation. Plus, it is noted that the biotin-present condition exhibited a stronger TurboID-csGBP signal than that in the biotin-absent condition. It seemed that the biotin favours to stabilize the free unbound TurboID-csGBP, especially for CC-PBX1 and VN-HOXA9 control conditions, which have a clear detectable HA signal, implying the potential non-specific binding between csGBP and one of the split halves (CC or VN) (α-HA channel, Figure II-9B). If so, surprisingly enough, only subtle PL can be detected in these negative controls, further justifying our former assumption, regarded as one of biotin side effects. proteasomes. To sum up, these results clearly demonstrate that GFP or GFP-derived BiFC can be effectively visualized and proximity-labeled using the BibID system at the presence of biotin in a native cell context.

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Figure II-9. Immunofluorescence of BibID test performed in HEK cells. Cells were fixed and stained with streptavidin-AlexaFluor555 (Strep-A555) to detect biotinylated proteins, and anti-HA antibody (α-HA) to detect ligase expression. BiFC/EGFP belongs to endogenous fluorescence. Nuclear DNA was labelled with DAPI stain. Non trasfected HEK cells as technical control. Scale bar, 20 μm. (A) Biotin-absent PL test.

(B) Biotin-present PL test. Exogenous 50uM biotin was prepared in complete culture medium and cells were treated for 10min before fixation.

Concerning the relative low efficiency of 3-plasmid transfection, we next substituted the BibID system with 2-plasmid system, leveraging a powerful bidirectional vector with Tet-responsive bidirectional promoter (Bi-PTRE) (Figure II-10A). In this new system, VN-HOXA9 and CC-PBX1 were assembled into the same plasmid, and cotransfected with a 2nd plasmid TurboID-csGBP in practice of BibID using. To assess its performance, the immunostaining test was similarly made in HEK cells, with or without biotin (Figure II-10B, 10C). As expected, in the presence of biotin, the BiFC-specific PL was obtained by 2-plasmid transfection, possessing the equivalent or better performance than previously.

This improvement is not only facilitating the transfection, but also benefit the further stable cell line making for true proteomics analysis.

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Figure II-10. Design of two-plasmid BibID system and performance test. (A) Illustration of 2-plasmid BibID system construction. All constructs were controlled by Tet-On system, driving the gene expression in presence of Dox. PTRE, Tet-responsive bidirectional promoter. Bi-PTRE, Tet-responsive bidirectional promoter. (B, C) In immunofluorescence test, after transfection, cells were fixed and stained with streptavidin-AlexaFluor555 (Strep-A555) to detect biotinylated proteins, and anti-HA antibody (α-HA) to detect ligase expression. BiFC/EGFP belongs to endogenous fluorescence. Nuclear DNA was labelled with

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