Split Fluorescent Protein Technology and (2) Present Five Case Studies How Chromotek’S Nano- Traps Can Be Applied to Tap the Full Potential of This Technology

Michael Metterlein, Christian Linke-Winnebeck ChromoTek GmbH, Am Klopferspitz 19, 82152 Planegg-Martinried, Germany

Abstract

To understand the biology of proteins and with that cell function in health and disease, it is crucial to be able to visualize proteins in situ and to analyze their protein-protein interaction networks in vivo. To this end, fluorescent proteins (FPs) have proven an invaluable tool as protein tags and have literally thrown light upon many scientific questions. Fluorescent proteins were further developed into split FP variants, spurred by the need for dedicated tools for the analysis of protein-protein interactions. Here, (1) we give a review about split fluorescent protein technology and (2) present five case studies how ChromoTek’s Nano- Traps can be applied to tap the full potential of this technology.

Summary

1. Jellyfish-derived GFP variants and most other fluorescent proteins from various species of coral or sea anemone share a common β-barrel fold composed of 11 single β-strands. Split fluorescent protein technology uses non-fluorescent fragments of this β-barrel, obtained by truncation between two or three β-strands to study single proteins or protein-protein interactions. Reconstitution of the full-length FP can be obtained either by conditioned (protein-protein interaction of fusion partners) or unconditioned (self- complementation) fragment association, recovering fluorescence. All aerobically grown cells and organisms that can be genetically modified have the potential to be used in split FP technology. 2. Split FP technology is frequently applied to cellular assays. ChromoTek’s Nano-Traps constitute attractive research tools for the biochemical validation of such experiments. For instance, the ChromoTek GFP-Trap has been successfully applied to different assay types such as protein self-complementation, BiFC, TriFC, or BiCAP, involving several different split GFP variants.

Introduction

A large number of fluorescent proteins Additionally, there are fluorescent proteins (FPs) has been discovered and developed that bind an exogenous chromophore (e.g. since the discovery of green fluorescent Halo, or Snap) but these are less frequently protein (GFP) in Aequorea victoria in 1962. used (Sanford and Palmer, 2017) and will Most FPs are derived from marine not be discussed here. For more details organisms such as jellyfish, coral, and sea about FPs in general, see also our anemone. These FPs bear an intrinsic informative ChromoBlog. chromophore.

www.chromotek.com 1

Figure 1. Comparison of wtGFP with seven of its variants (eGFP, sfGFP, eYFP, Venus, BFP, CFP and eCFP) and two GFP homologs from other organisms (mCherry and mNeonGreen). A: Structural alignment of all selected FPs (except CFP), shown as cartoon presentation with the chromophore drawn as orange sticks in the center of the β-barrel. Color code and PDB IDs are listed in B. The structures were aligned using PDBeFold (http://www.ebi.ac.uk/msd-srv/ssm/) and adapted using PyMOL B: Overview of the selected FPs showing their mutations compared to wtGFP. C: Sequence identities in [%] between the selected FPs. “mNG” is mNeonGreen. The protein sequences were retrieved from http://www.fpbase.org, and sequence identities were calculated using the software Geneious.

A vast number of fluorescent proteins has (confirmed by an overall root mean square been structurally characterized. Figure 1 deviation (rmsd) of only ~1.2 Å over 207 gives an overview of ten such fluorescent compared backbone Cα atoms). In proteins that have been selected because contrast, the shared sequence identity of their published use in split FP assays may be as low as 20-30 % (Figure 1C). (see above and Table 1). In practical terms, the sequence variety of These jellyfish-derived GFP variants (first fluorescent proteins derived from different eight in Figure 1B), mCherry from a sea organisms means that most anti-GFP anemone, and mNeonGreen from a antibodies exclusively bind to various sets lancelet share a highly conserved β-barrel of GFP variants derived from jellyfish. fold with a chromophore in its center. This Other FP homologs such as mCherry or chromophore is formed by three mNeonGreen require other, dedicated consecutive amino acids at positions 65, tools. ChromoTek offers such research 66, and 67 in an autocatalytical process tools for fluorescent proteins derived from called maturation. many different species.

The high structural conservation between Protein-Protein Interaction Assays the ten selected FPs is illustrated by the Since the discovery of GFP, fluorescent structural alignment in Figure 1A proteins have become wildly successful

2 www.chromotek.com

tools in biological and biomedical research, partners, the fragments of the reporter as recognized by the Nobel Prize in protein are brought into close proximity chemistry in 2008. The tagging of a protein and are thus able to reconstitute an active of interest (POI) by fusing it to a FP is widely protein. The reconstitution of a reporter used to visualize a POI’s subcellular protein from its fragments is also termed location in vivo or in vitro, to study its protein complementation. However, biochemistry (using methods such as protein complementation cannot only be immunoprecipitation), or to analyze mediated in a conditioned way by fluorescent cells by flow cytometry interacting fusion proteins (as just (Leonetti et al., 2016). described above), but also in an unconditioned way (self- Usually, it is not the single protein in complementation). The binding route isolation that is of interest, but rather its highly depends on the kind of split FP network of interactions with other fragments used. proteins. These protein-protein interactions (PPI) are fundamental to all The basic principle of protein processes of life, e.g. in signal transduction complementation can be traced back to or gene expression. Many methods have the 1950s, when Richards observed that been developed to study protein-protein subtilisin-cleaved fragments of interactions in cells, some of which are ribonuclease are active again after self- based on or can be used with (split) FPs. complementation (Kerppola, 2008). Since then, various protein complementation One of the most frequently applied assays using fragments of reporter methods is Förster resonance energy proteins such as β-galactosidase, TEV transer (FRET). It allows real-time detection protease, or luciferase have been of complex formation and dissociation. developed. However, the method has low sensitivity and works only if the fluorescent reporter However, these reporter proteins are not proteins are placed within 10 nm of each optimal for usage in protein-protein other (Kerppola, 2008, Miller et al., 2015). interaction assays, because the observed reporter enzyme products diffuse away Another PPI method is ChromoTek’s from the investigated protein-protein Fluorescent Two-Hybrid (F2H®) assay. This interaction site. This diffusion leads to assay enables real-time monitoring and impaired colocalization of marker activity quantitative analysis of interactions and PPI site (Kerppola, 2008, Cabantous et between GFP- and RFP-tagged proteins in al., 2013). By contrast, the use of live mammalian cells. fluorescent protein fragments as tags in Protein Complementation Assays protein complementation assays, first described by Ghosh and coworkers Protein-protein interactions can also be (Ghosh et al., 2000), enables studies with analyzed using protein complementation the marker activity inherent to the PPI site. assays. These assays all rely on the use of Ghosh and colleagues dissected a GFP fragments of a reporter protein, e.g. an variant (sg100GFP) between β-strands 7 enzyme or FP, that are fused to interacting and 8 and fused the resulting fragments to proteins. Upon interaction of the fusion strongly interacting antiparallel leucine www.chromotek.com 3

zippers. They revealed that reconstitution In 2003, Hu and Kerppola published of this split GFP variant is driven only by the designs for further variants of split FPs, interaction of both leucine zippers, works which were intended for the use in in vivo (e.g. E.coli) and in vitro (after GFP (multicolor) BiFC (Hu and Kerppola, 2003). fragment purification from inclusion They generated fragments of eGFP, eYFP, bodies), and that absorption and emission eCFP, and eBFP truncated either between maxima are identical to parental full-length β-strands 7 and 8 or between β-strands 8 GFP (Ghosh et al., 2000, Kerppola, 2008). and 9 (Figure 2). The resulting fragments and combinations thereof were tested as Progress in Split Fluorescent Protein fusions with interacting leucine zippers. Technology Several combinations of fragments yielded In the following, we will describe key indeed detectable fluorescence signals, developments in the field of split FP assays. but displayed varying tendencies for self- For an overview, commonly used split FPs complementation. Such self-binding is not are listed in Table 1; their split sites are desired in BiFC assays and can be mapped to a topology diagram of GFP in minimized by reducing expression of the Figure 2. fragment fusions to the lowest level that permits fluorescence detection. In the early 2000s, Hu and coworkers identified the first eYFP fragments that While split eYFP had been suggested for form active complements only upon usage in BiFC assays by Kerppola in 2008, binding of their interacting fusion partners. the eYFP variant Venus is recommended as They termed this mechanism of the split FP variant to use in BiFC assays by conditional complementarity “bimolecular several authors today. (Kerppola, 2008, fluorescence complementation” (BiFC) (Hu Miller et al., 2015, Zeng et al., 2017). et al., 2002).

Figure 2. Secondary structure topology diagram of GFP and its variants. Shown are the 11 β-strands of GFP (or its variants) and the central α-helix. Black numbers on β-strands indicate the number of the respective β-strand, whereas the grey numbers on top or at the bottom of a β-strand mark individual amino acids (aa). The chromophore of any GFP variant is shown as a green star in the middle of the α-helix. Additional stars in red, blue, black and yellow mark split sites between β-strands for different split FP variants (see Table 1 for colour codes).

4 www.chromotek.com

Table 1. Overview of selected split fluorescent proteins. Selection was based on estimated relevance of split FPs. Numbers after each split FP variant refer to respective β- strands included in fragments. Colored stars refer to split sites in Figure 2.

Split FP Split (sg100) GFP Tripartite Split GFP Split eYFP * Split Venus Split eCFP * Split mLumin Split (sf) GFP Split mNG2 Split sfCherry2 1-7/8-11 1-9/10/11 1-7/8-11 1-7/8-11 1-7/8-11 1-7/8-11 1-10/11 1-10/11 1-10/11 N-term fragment GFP1-7 GFP1-9 eYFP1-7 Venus1 eCFP1-7 mLumin1-7 GFP1-10 opt mNG2 1-10 sfCherry2 1-10 C-term fragment(s) GFP8-11 GFP10, GFP11 eYFP8-11 Venus2 eCFP8-11 mLumin8-11 GFP11 (M3) mNG2 11 sfCherry2 11 Based on: wtGFP sfGFP eYFP eYFP --> seYFP eCFP tagRFP sf GFP mNeonGreen mCherry --> --> Venus sfCherry Split FP Mutations: GFP1-7: F64L, GFP1-9 opt: S2R, V16E, (no mutations) (no mutations) (no mLumin1-7: GFP1-10 opt: N39I, mNG2 1-10: sfCherry2 1-10: S65C, Q80R, Y151L S28F, N39I, T43S, S99Y, mutations) R67K, N143S T105K, E111V, K128M, S142T, E118Q, T128I GFP8-11: I167T, N149K, K158N, K166T mLumin8-11: I128T, K166T, R150M, G172V sfCherry2 11: K238N GFP10 (T10): L194D, S158A, F174L, I167V, S205T mNG2 11: G219A N198D, S205T, V206I, H197R GFP11 (M3): V228M P211L L221H, F223Y, GFP11 (S11): F223Y, T225N insertion: DAS231-233 Split site(s) (aa) 157/158 193/194 and 212/213 154/155, 154/155 154/155 151/152 214/215 214/215 207/208 (173/174) ** (173/174) ** Applications *** BiFC TriFC BiFC BiFC, BiCAP BiFC BiFC Self-complemen- Self-complemen- Self-complementation tation tation

Research tools a-GFP VHH as a-GFP VHH a-GFP VHH a-GFP VHH as none **** a-RFP V HH as a-GFP VHH as a- mNeonGreen a-RFP VHH as

offered by used in GFP-Trap as used in GFP-Trap as used in GFP- used in GFP-Trap used in RFP- used in GFP-Trap VHH as used in used in RFP-Trap ChromoTek (predicted) (e.g. Foglieni et al Trap (predicted) (e.g. Trevelyan et Trap (e.g. Leonetti et mNeonGreen- (predicted) 2017) al 2019) (predicted) al 2016) Trap (predicted)

Ex/Em (nm) 485/510 485/510 500/535 500/530 436/470 587/621 485/510 500/520 590/610

Reference Ghosh et al 2000 Cabantous et al 2013 Hu et al 2002, Trevelyan et al. Hu et al 2003 Miller et al Cabantous et al Feng et al 2017 Kamiyama et al Hu et al 2003 2019 2015 2005 2016, Feng et al 2017 Origin Aequorea victoria Aequorea victoria Aequorea victoria Aequorea victoria Aequorea Entacmea Aequorea victoria Branchiostoma Discosoma sp. victoria quadricolor lanceolatum

*Split eYFP and split eCFP show poor maturation rates at 37°C (Miller et al., 2015); **This split site is functional, but site 154/155 is recommended, generally; ***Only successfully tested applications are listed (not exhaustive); ****Only weak binding of GFP-Trap due to N146I mutation in eCFP (Rothbauer et al., 2008);

www.chromotek.com 5

In 2005, the Waldo laboratory developed a mNeonGreen2 or red sfCherry2 were split GFP variant optimized for self- introduced by Feng and coworkers (Feng et association of its fragments and minimal al., 2017). Using these variants, the authors fusion partner disturbance. This split FP created two-color or super-resolution system is not intended to be applied to PPI images of endogenous proteins. Red, far- studies, but to high-throughput solubility red and near-infrared split FPs have been screens of libraries of protein mutants. In the focus of further research efforts. contrast to the split GFP variants of Ghosh However, the current red fluorescent or Hu, Cabantous’ split variant was based protein (RFP) variants, split mCherry and on superfolder (sf) GFP, which was split mRFP1 (Q66T), are functional only at truncated between β-strand 10 and 11. N- relatively low temperatures (<30°C). and C-terminal fragments both were In the far-red range, two mutants of mKate, mutated to optimize their suitability for mLumin (Ex: 587 nm/Em: 621 nm, Table 1) fluorescence complementation and to and Neptune (Ex: 600 nm/Em: 650 nm) reach high solubility (Cabantous et al., seem to be suitable for BiFC assays also at 2005). 37°C. Even longer excitation maxima A few years later, the Waldo laboratory (690 nm) can be targeted with the near- described an entirely new interaction assay infrared FP iRFP, a bacterial phytochrome based on tripartite split GFP (referred to as unrelated to the FPs discussed so far, tripartite fluorescence complementation, which was successfully tested in BiFC TriFC) (Cabantous et al., 2013). In TriFc, assays (Miller et al., 2015). three fragments are created by the double It would exceed the scope of this dissection of GFP between β-strands 9 and whitepaper to describe all the recent 10, and between β-strands 10 and 11, advances in the field of split fluorescent respectively. The advantage of TriFC over proteins. Notwithstanding, we will describe BiFC consists in the small size of the some specific applications in more detail in resultings tags, GFP10 (19 aa) and GFP11 the applications part below. (21 aa), which can be complemented by GFP1-9. GFP10 and GFP11 are also called Assay Types Based on Split FP Variants T10 and S11 and are optimized for N- As already mentioned, the split FP variants terminal tagging of a fusion partner. It was discussed above can be applied to three shown that fluorescence complementation basic assay types, FP self- only occurs upon interaction of the fusion complementation, bimolecular partners of GFP10 and GFP11, rendering complementation and tripartite FP TriFC a promising tool to study PPIs in vitro complementation. Here, we will and in vivo. In addition to PPI studies, TriFC summarise the basic characteristics of allows the screening of libraries of these assay classes: interacting proteins or of libraries of small compounds inhibiting PPIs. FP self-complementation assays use FP fragments that self-associate To diversify the range of colors of split FPs, independently of any fusion partner additional variants based on yellow-green (Figure 3A).

6 www.chromotek.com

Figure 3. Schematic representation of fluorescent protein complementation assays. FN is the N-terminal and FC1/2 the C- terminal fragment(s) of a fluorescent protein (FP). X and Y are interacting proteins fused to fragments of a FP. A: Fluorescent protein self-complementation, B: Bimolecular fluorescent complementation (BiFC), C: Tripartite fluorescent complementation (TriFC).

C-terminal fragments of FPs used in self- between the fusion proteins. complementation assays are generally Subsequently, the C-terminal fragments smaller than those used in BiFC. The main (FC1 and FC2) associate, but will only emit aim of self-complementation assays is to fluorescence upon binding of the third study a single POI, e.g. its expression level, fragment, the N-terminal FN, which and not its interaction network. reconstitutes the full FP.

The principle of bimolecular fluorescent By using BiFC or TriFC, PPIs can be protein complementation (BiFC) is shown visualized in cells or in vitro. For both in Figure 3B. A fluorescent protein (FP) is assays, minimal background fluorescence split into two parts, FN and FC, both fused of the FP fragments in the absence of to interacting proteins X and Y. Ideally, the interacting fusion partners is essential, FP complementation is driven only by the which translates to a minimum in self- interaction of both fusion proteins. In such association. an optimal case, X and Y bind to each other All three assay formats have in common and thus bring their FP fragments into that the reconstitution of FP fragments is proximity. Consequently, fragment virtually irreversible. For BiFC and TriFC reconstitution occurs quite fast and is assays, this means that even weak or virtually irreversible. Eventually, the transient protein interactions can be fluorophore forms via maturation and detected (Cabantous et al., 2013, Foglieni gives rise to a fluorescence signal (Foglieni et al., 2017, Kerppola, 2008). et al., 2017, Kerppola, 2008). The main aim of BiFC is the analysis of protein-protein Combining split FP with VHH Technology interactions in vivo. Split FPs enable the visualization of PPIs, Tripartite fluorescent protein which can then be validated using complementation (TriFC, Figure 3C) biochemical methods, e.g. (co-) requires the splitting of an FP into three immunoprecipitation using FP-binding parts (FN, FC1 and FC2). Both interacting antibodies. In recent years, a remarkable proteins (X, Y) are tagged with one of the class of single-domain antibodies called small C-terminal fragment tags, FC1 or FC2 VHHs (or nanobodies) has emerged as an (~20 aa) (Cabantous et al., 2013). Small tags ideal tool for immunoprecipitation and can be advantageous for some target other biochemical assays. VHHs constitute proteins as they are expected to exercize the heavy chain variable domain derived minimal influence on the tagged protein. from camelid heavy chain-only antibodies As in BiFC, the initial binding event occurs and are among the smallest known www.chromotek.com 7

functional antibody fragments (13-15 kDa). Foglieni et al. 2017 – an example for TriFC

Like conventional antibodies, VHHs bind to Foglieni and coworkers employed split GFP their cognate antigen with high affinity, i.e. to structurally characterize and quantify with dissociation constants (KD) in the functional biomolecular interactions in the nanomolar to low picomolar range. Unlike context of neurodegeneration most conventional antibodies, however, (frontotemporal dementia (FTD)) (Foglieni VHHs tend to be extremely stable and et al., 2017). The investigation of how, remain functional at high temperatures when, and where certain proteins (self-) and under harsh chemical conditions (van interact to form possibly toxic aggregates der Linden et al., 1999, Dumoulin et al., is crucial to better understand 2002, Muyldermans, 2013). neurodegeneration. As model proteins, Of special interest is ChromoTek’s GFP- they used Tau and TAR-DNA-binding

Trap, which comprises an anti-GFP VHH protein (TDP-43), which are both immobilized to agarose-beads for one- associated with FTD. step immunoprecipitation. The By using TriFC, they showed that the ChromoTek GFP-Trap binds to full-length trimolecular complex of GFP10-TDP-43, GFP and GFP derivatives like eGFP, sfGFP, GFP11-TDP43 and GFP1-9 reflects the eYFP, Venus, CFP (less so eCFP) or BFP with subcellular localization of nuclear TDP-43 very high affinity (dissociation constant KD assemblies. They also successfully stained of 1 pM1). In the context of split GFP or its post-fixed cells, co-transfected with variants, it captures only fully reconstituted GFP10-TDP-43 and GFP11-TDP-43, by the GFP, but not its fragments, which enables simple addition of recombinantly the combination of BiFC with produced GFP1-9. This experiment immunoprecipitation (see below). confirmed that TDP-43 self-assembly Case studies of the application of occurs independently of the presence of ChromoTek’s GFP-Trap to split FP GFP1-9. experiments Using the ChromoTek GFP-Trap Magnetic ChromoTek’s GFP-Trap has been cited in Agarose (gtma), they validated these more than 1800 publications for the use in protein-protein interactions biochemically. immunoprecipitation, a number of which Lysates of HEK293 cells, co-transfected also refer to split FPs. In the following, we with GFP10-HA-TDP-43, GFP11-β1-TDP-43 will present five case studies, in which and GFP1-9, were analyzed by ChromoTek’s GFP-Trap was used together immunoprecipitation and Western Blot with split FPs. Common to these five (IP/WB). Again, they observed interaction studies is that each add an innovative between the two TDP-43 constructs in the approach to the field of split FPs, presence of GFP1-9. If any of the three emphasizing how this field is still evolving. components was missing, no protein was precipitated, which underlines the

1 Kinetic parameters of the GFP-Trap have been determined using Dynamic Biosensors’ switchSENSE® technology

8 www.chromotek.com

specificity of the ChromoTek GFP-Trap for established multiprotein complexes, the fully reconstituted split GFP. namely cohesin, SEC61 translocon, clathrin, and SPOTS sphingolipid synthesis Leonetti et al. 2016 – an example for self- complex. For each complex, a single complementation subunit had been tagged using GFP11 and Leonetti and colleagues combined was used as bait-protein for the CRISPR/CAS9 and split GFP technology immunoprecipitation using ChromoTek (GFP1-10 & GFP11) to tag endogenous GFP-Trap. Western blot analysis confirmed human genes (Leonetti et al., 2016). They the presence of the bait as well as of its used HEK293T cells stably expressing expected interactions partners. GFP1-10 as a parental cell line. Using Moreover, they modified this strategy to CRISPR/CAS9 technology, they generated enable the specific and non-denaturing 48 cell lines by fusing GFP11 to different release of captured proteins from the endogenous model proteins, representing ChromoTek GFP-Trap. Non-denaturing various subcellular locations such as the elution is desired for subsequent activity cytoskeleton, endoplasmic reticulum, assays or structural studies, for example. nucleus, or endosomes. To this end, they included a TEV protease Crucially, the small size of the 16-aa GFP11 cleavage site between the GFP11 tag and tag enabled the authors to transduce the the POI. Thus, captured target protein was parental cell line with single-guide eluted using on-resin TEV protease RNA/CAS9 ribonucleoprotein complexes. cleavage, which yielded protein of high In 30 of 48 cases, the resulting purity – despite the low abundance of the fluorescence signal was sufficiently high to endogenous proteins in question. This be detected by flow cytometry analysis. In example underlines the unusually high additional cases, successful albeit low affinity of the ChromoTek GFP-Trap, which expression of GFP11-tagged protein was results in very efficient pulldown observed using confocal microscopy. The experiments even for proteins of low overall knock-in efficiency (i.e. the fraction expression. of green fluorescent cells) was determined Croucher et al. 2016 – combining BiFC with to be approximately 36% using flow immunoprecipitation (BiCAP) cytometry. The fluorescence signal could be increased using repeats of GFP-11 tags Croucher and coworkers introduced a new owing to a higher number of self- method, which they dubbed bimolecular complemented GFP molecules. In addition, complementation affinity purification GFP self-complementation allowed the (BiCAP). They combined conformation- correlation of the GFP signal to protein specific nanobodies with a protein- expression levels by ribosome profiling. fragment complementation assay and affinity purification (Croucher et al., 2016). In a next step, Leonetti et al. illustrated the Traditionally, affinity purification coupled benefit of their GFP1-10/GFP-11 with tandem mass spectrometry (AP- complementation approach for the MS/MS) is used to isolate a single bait analysis of native, endogenous networks. protein and its interaction partners. In Using the ChromoTek GFP-Trap Agarose contrast, BiCAP facilitates the specific (gta), they isolated four native, well- www.chromotek.com 9

isolation and characterization of the Thus, Trevelyan and colleagues set out to interactome of a binary protein complex. In further investigate the formation of these their report, the interactome of ERBB2 signalosomes. (also known as Her2), a member of the In one of their experiments, they family of epidermal growth factors (EGFR), determined the stoichiometry of the was characterized either in the form of a complex comprising ASK1 and ASK2. Using homo- or a heterodimer (with EGFR or the ChromoTek GFP-Trap Agarose (gta), ERBB3). The ERBB2 gene is amplified in they immunoprecipitated two split Venus many breast cancer cells, and ERBB2 fusion proteins, ASK1-Venus1 and ASK1- dimers are targeted by several therapeutic Venus2, from HEK293T cells followed by agents. mass spectrometry analysis. Although The authors used split Venus (a variant of ASK1 was overexpressed, the abundance GFP) and specifically selected the of ASK2 was 75% of ASK1, which indicates ChromoTek GFP-Trap for a selective incorporation of near-equal immunoprecipitation analysis, as the GFP- ratios of ASK1 and ASK2. In addition, other Trap captures only reconstituted Venus, proteins assumed to interact with ASK1 i.e. the ERBB2 homo-/heterodimers. MS were identified, too (e.g., ASK3 or several analysis of BiCAP-isolated receptor dimers members of the ubiquitin ligase family). As revealed a core interactome of ten protein for Croucher et al., the key to this for the three dimer pairs (ERBB2/ERBB2, experiment was the use of the ChromoTek ERBB2/EGFR, ERBB2/ERBB3), but identified GFP-Trap, which only binds to also a set of proteins distinct to each reconstituted Venus protein and not to dimer. Thus, the BiCAP approach in Venus1 or Venus2 fragments, facilitating combination with the ChromoTek GFP- interactome studies of ASK1 dimers only. Trap provides a powerful method for the Dáder et al. 2019 analysis of interactomes. Using the split GFP system GFP1-10/11, Trevelyan et al. 2019 – another example of Dáder and coworkers (Dáder et al., 2019) BiCAP have conducted immunoprecipitation Trevelyan et al. (Trevelyan et al., 2019) experiments with Aradopsis thaliana and a applied the BiCAP approach and split plant virus protein. In this specific case, P6 Venus to apoptosis signal-regulating protein, also termed transactivator- kinases (ASK1-3), which are activators of viroplasmin (TAV), a key player in the viral the P38 and JNK MAP kinase pathways. replication cycle from Cauliflower mosaic ASK1, for example, is associated with virus was used (CaMV). As CaMV is a plant melanoma, gastric cancers, or non- virus with a small circular DNA genome of alcoholic steatohepatitis (NASH). Several 8 kb that does not tolerate genome inhibitors of ASK1 are the object of clinical insertions longer than a few hundred trials. The kinases ASK1-3 form oligomeric nucleotides, tagging of P6 with the 16 complexes called ASK signalosomes, a amino acid GFP fragment GFP11 (termed process, which is not fully understood yet. 11P6) was key to facilitate these studies.

10 www.chromotek.com

GFP1-10 transgenic Aradopsis thaliana GFP-Trap exclusively binds to the

GFP1-10 was infected with CaMV11P6. Plants reconstituted form of the split GFP variant developed typical mosaic, yellowing and Venus. This binding mechanism is stunting symptoms like control plants predicted to hold true for other split GFP inoculated with CaMVwt, however, a little variants with similar split sites. time-delayed. GFP reconstitution of While there are no data available yet for CaMV11P6-infected A. thaliana GFP1-10 non-GFP-derived split fluorescent proteins plants was successfully confirmed by such as mNeonGreen2, sfCherry2 or whole plant imaging with a fluorescence mLumin, other ChromoTek NanoTraps, scanner. e.g. mNeonGreen-Trap or RFP-Trap, may Cell lysates prepared from healthy and also bind their reconstituted target FP and

CaMV11P6-infected A. thaliana GFP1-10 thus further facilitate biochemical leaves were subjected to experiments. In addition to the use of immunoprecipitation using the ChromoTek’s Nano-Traps, other VHH ChromoTek GFP-Trap Magnetic Agarose formats might be applicable to split FP (gtma). Subsequent SDS-PAGE and assays. For instance, one could envisage Western blot analysis showed that 11P6 the use of unconjugated ChromoTek a-FP was indeed captured by the ChromoTek VHH in sandwich immunoassays or of the GFP-Trap, indicating affinity-purification of ChromoTek Nano-Boosters in 11P6 as part of the reconstituted split GFP immunofluorescence experiments with complex. split FP variants.

Conclusion References

Split fluorescent protein complementation CABANTOUS, S., NGUYEN, H. B., PEDELACQ, J.-D., KORAÏCHI, F., CHAUDHARY, A., GANGULY, assays are broadly applicable to the K., LOCKARD, M. A., FAVRE, G., visualization of target proteins or protein- TERWILLIGER, T. C. & WALDO, G. S. 2013. A protein interactions within their cellular new protein-protein interaction sensor setting, to the screening of libraries, or to based on tripartite split-GFP association. Scientific reports, 3, 2854-2854. cell sorting. In conjunction with other CABANTOUS, S., TERWILLIGER, T. C. & WALDO, G. S. biochemical methods (e.g. 2005. Protein tagging and detection with immunoprecipitation and mass engineered self-assembling fragments of green fluorescent protein. Nat Biotechnol, spectrometry) they are very powerful 23, 102-7. orthogonal validation methods. CROUCHER, D. R., ICONOMOU, M., HASTINGS, J. F., KENNEDY, S. P., HAN, J. Z., SHEARER, R. F., As underlined by our small selection of MCKENNA, J., WAN, A., LAU, J., APARICIO, S. case studies (Foglieni et al., 2017, Leonetti & SAUNDERS, D. N. 2016. Bimolecular complementation affinity purification et al., 2016, Trevelyan et al., 2019, Dáder et (BiCAP) reveals dimer-specific protein al., 2019, Croucher et al., 2016), VHH-based interactions for ERBB2 dimers. Sci Signal, 9, reagents such as the ChromoTek GFP-Trap ra69. DÁDER, B., BURCKBUCHLER, M., MACIA, J.-L., ALCON, are highly valuable tools in split FP assays. C., CURIE, C., GARGANI, D., ZHOU, J. S., NG, For example, in a bimolecular J. C. K., BRAULT, V. & DRUCKER, M. 2019. complementation and affinity purification Split green fluorescent protein as a tool to study infection with a plant pathogen, assay (BiCAP), it was shown by Croucher et Cauliflower mosaic virus. PLOS ONE, 14, al. and Trevelyan et al. that the ChromoTek e0213087. www.chromotek.com 11

DUMOULIN, M., CONRATH, K., VAN MEIRHAEGHE, A., LEONHARDT, H. 2008. A Versatile Nanotrap MEERSMAN, F., HEREMANS, K., FRENKEN, L. for Biochemical and Functional Studies with G. J., MUYLDERMANS, S., WYNS, L. & Fluorescent Fusion Proteins. Molecular MATAGNE, A. 2002. Single-domain & Cellular Proteomics, 7, 282. antibody fragments with high SANFORD, L. & PALMER, A. 2017. Chapter One - conformational stability. Protein science : a Recent Advances in Development of publication of the Protein Society, 11, 500- Genetically Encoded Fluorescent Sensors. 515. In: THOMPSON, R. B. & FIERKE, C. A. (eds.) FENG, S., SEKINE, S., PESSINO, V., LI, H., LEONETTI, M. Methods in Enzymology. Academic Press. D. & HUANG, B. 2017. Improved split TREVELYAN, S. J., BREWSTER, J. L., BURGESS, A. E., fluorescent proteins for endogenous CROWTHER, J. M., CADELL, A. L., PARKER, B. protein labeling. Nat Commun, 8, 370. L., CROUCHER, D. R., DOBSON, R. C. J., FOGLIENI, C., PAPIN, S., SALVADE, A., AFROZ, T., MURPHY, J. M. & MACE, P. D. 2019. PINTON, S., PEDRIOLI, G., ULRICH, G., Mechanism of preferential complex POLYMENIDOU, M. & PAGANETTI, P. 2017. formation by Apoptosis Signal-regulating Split GFP technologies to structurally Kinases. bioRxiv, 693663. characterize and quantify functional VAN DER LINDEN, R. H. J., FRENKEN, L. G. J., DE GEUS, biomolecular interactions of FTD-related B., HARMSEN, M. M., RUULS, R. C., STOK, W., proteins. Sci Rep, 7, 14013. DE RON, L., WILSON, S., DAVIS, P. & GHOSH, I., HAMILTON, A. D. & REGAN, L. 2000. VERRIPS, C. T. 1999. Comparison of physical Antiparallel Leucine Zipper-Directed chemical properties of llama VHH antibody Protein Reassembly: Application to the fragments and mouse monoclonal Green Fluorescent Protein. Journal of the antibodies. Biochimica et Biophysica Acta American Chemical Society, 122, 5658-5659. (BBA) - Protein Structure and Molecular HU, C.-D., CHINENOV, Y. & KERPPOLA, T. K. 2002. Enzymology, 1431, 37-46. Visualization of Interactions among bZIP ZENG, L., WANG, W. H., ARRINGTON, J., SHAO, G., and Rel Family Proteins in Living Cells Using GEAHLEN, R. L., HU, C. D. & TAO, W. A. 2017. Bimolecular Fluorescence Identification of Upstream Kinases by Complementation. Molecular Cell, 9, 789- Fluorescence Complementation Mass 798. Spectrometry. ACS Cent Sci, 3, 1078-1085. HU, C.-D. & KERPPOLA, T. K. 2003. Simultaneous visualization of multiple protein Disclaimer interactions in living cells using multicolor fluorescence complementation analysis. For research use only. Nature biotechnology, 21, 539-545. KERPPOLA, T. K. 2008. Bimolecular fluorescence complementation (BiFC) analysis as a probe ChromoTek’s products and technology are covered of protein interactions in living cells. Annu by granted patents and pending applications owned Rev Biophys, 37, 465-87. by or available to ChromoTek GmbH by exclusive LEONETTI, M. D., SEKINE, S., KAMIYAMA, D., license. ChromoTek, Chromobody, F2H, GFP-Trap, WEISSMAN, J. S. & HUANG, B. 2016. A Myc-Trap, RFP-Trap, Spot-Tag, Spot-Label, and Spot- scalable strategy for high-throughput GFP Trap are registered trademarks of ChromoTek tagging of endogenous human proteins. GmbH. Nanobody is a registered trademark of Proceedings of the National Academy of Ablynx, a Sanofi company. SNAP-tag is a registered Sciences, 113, E3501-E3508. trademark of New England Biolabs, Inc. HaloTag is a MILLER, K. E., KIM, Y., HUH, W.-K. & PARK, H.-O. 2015. registered trademark of Promega Corporation. Bimolecular Fluorescence Other suppliers’ products may be trademarks or Complementation (BiFC) Analysis: registered trademarks of the corresponding Advances and Recent Applications for supplier each. Statements on other suppliers’ Genome-Wide Interaction Studies. Journal products are given according to our best knowledge. of Molecular Biology, 427, 2039-2055. MUYLDERMANS, S. 2013. Nanobodies: Natural Single-Domain Antibodies. Annual Review of Biochemistry, 82, 775-797. ROTHBAUER, U., ZOLGHADR, K., MUYLDERMANS, S., SCHEPERS, A., CARDOSO, M. C. &

12 www.chromotek.com