Spy Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox AbstractPeptide tags are a key resource, introducing minimal change while enabling a consistent process to purify diverse proteins. However, peptide tags often provide minimal benefit post-purification. We previously designed SpyTag, forming an irreversible bond with its protein partner SpyCatcher. SpyTag provides an easy route to anchor, bridge or multimerize proteins. Here we establish Spy Go, enabling protein purification using SpyTag. Through rational engineering we generated SpyDock, which captures SpyTag-fusions and allows efficient elution. Spy Go enabled sensitive purification of SpyTag-fusions from Escherichia coli, giving superior purity than His-tag/nickel-nitrilotriacetic acid. Spy Go allowed purification of mammalian-expressed, N-terminal, C-terminal or internal SpyTag. As an oligomerization toolbox, we established a panel of SpyCatcher-linked coiled coils, so SpyTag-fusions can be dimerized, trimerized, tetramerized, pentamerized, hexamerized or heptamerized. Assembling oligomers for Death Receptor 5 stimulation, we probed multivalency effects on cancer cell death. Spy Go, combined with simple oligomerization, should have broad application for exploring multivalency in signaling. IntroductionAffinity chromatography is a central enabling technology for research and for producing therapeutics, vaccines, and diagnostics1,2. However, a persistent problem with affinity tags is paradoxically the tags themselves, since the tags often serve no purpose post-purification. Tags can inhibit crystallization3, interfere with protein interactions4, and produce an unhelpful immune response in vivo5. Tags may be removed by proteolysis but this extra step is time-consuming, often inefficient and reduces overall yield6,7.There are already a multitude of affinity tags1,8,9,10,11. However, each tag presents its own limitations. The most widely used, the His-tag, is not without faults. There are many examples of His-tagging disrupting protein solubility12, structure13,14, and function15,16. His-tag purification faces particular challenges from leakage of metal ions into downstream assays17,18 and substantial immunogenicity5. The four-amino acid C-tag is less immunogenic but is only functional at the C-terminus8. Apart from purification, it would be desirable to use peptide tags for assembly or immobilization, but the low stability of peptide interactions is frequently limiting19,20.We previously developed a peptide-protein pair, SpyTag and SpyCatcher, that spontaneously forms a covalently-linked complex21,22 (Fig.聽1). We continued the progress by increasing the rate of reaction via the evolved SpyTag002 and SpyCatcher002 versions23. SpyTag technology has enabled diverse applications including Plug-and-Display vaccine assembly24,25,26, multivalent activation of signaling27, modular antibody decoration28, and living or catalytic biomaterials29,30,31,32. Up until now, Spy proteins were nearly always purified via a His-tag, which required further tag cleavage for immunological applications24. Here, we report the development of an affinity chromatography technique employing SpyTag as a purification tag, by step-wise engineering of the SpyCatcher protein partner (Fig.聽1). In addition, to extend the modular expansion of protein function, we establish a toolbox for oligomerization of proteins of interest using SpyTag-mediated covalent reaction. These oligomers shed light on the nature of multivalent signaling of DR5 to induce cancer cell killing.Fig. 1Overview of Spy Go. SpyTag/SpyCatcher interact irreversibly by spontaneous isopeptide bond formation, promoted by E77. SpyTag purification requires the generation of reversible interaction with SpyDock in 3 steps: blocking reaction, enhancing efficiency of SpyTag binding and precise immobilization on resin. Spy Go then enables purification of SpyTag-fusions, setting the stage for modular oligomerization or multimerizationFull size imageResultsEstablishment of the Spy Go purification systemAs a first step to establish Spy Go purification, the formation of an isopeptide bond between SpyCatcher and SpyTag must be abrogated, to make possible the elution of SpyTag-fusions. To generate a non-reactive 鈥減seudo-SpyCatcher鈥? the activating glutamic acid residue in the CnaB2 triad (E77, Fig.聽1) was mutated to aspartic acid to retain the charge, or to alanine, glycine, asparagine, glutamine, serine, threonine, or valine to remove any possibility of proton donation/acceptance33. We mixed SpyTag-MBP and the E77X variants at 25鈥壜癈 for 24鈥塰. E77D still showed a small amount of reaction with SpyTag-MBP, but no trace of reaction was seen with any other mutation (Fig.聽2a).Fig. 2Design of SpyDock. a All small residues except D blocked SpyCatcher reaction. E77 was mutated to the indicated residue and incubated with SpyTag-MBP for 24鈥塰, before boiling in SDS and analysis by SDS-PAGE with Coomassie staining. b Additional mutations in SpyCatcher enhanced Spy Go purification. The original SpyCatcher, the evolved SpyCatcher002 or SpyCatcher2.1 bearing the E77A mutation were compared for capture and elution of SpyTag-MBP (SDS-PAGE with Coomassie staining). Further E77X mutations were similarly explored on SpyCatcher2.1. c Positions of mutations screened for SpyDock acceleration, based on Protein Data Bank 4MLI; CnaB2 triad represented in spheres, anchoring sites in yellow, and SpyCatcher2.1 accelerating mutations in green. d S49C resin anchoring allowed efficient SpyDock purification. SpyCatcher2.1 E77A S49C was coupled to SulfoLink resin and tested for SpyTag-MBP capture and release (SDS-PAGE with Coomassie staining)Full size imageTo advance Spy Go purification further, we hypothesized that the rate of association between SpyTag and the SpyCatcher moiety could be limiting. We have previously generated SpyCatcher002 through phage display evolution for enhanced interaction with SpyTag23. Here we additionally increased the negative surface charge on SpyCatcher and reduced the flexibility in one of the loops through proline substitution, generating SpyCatcher2.1. When purified SpyTag-MBP was incubated with the different E77X variants, SpyCatcher002 led to more efficient capture and elution than SpyCatcher, while SpyCatcher2.1 was more effective still (Fig.聽2b). Comparing alternative E77 mutations for SpyCatcher2.1, E77A led to recovery of the highest amount of SpyTag-MBP (Fig.聽2b). Hence, this mutant was taken forward for subsequent development.We aimed to attach SpyCatcher2.1 E77A site-specifically to sepharose beads, for maximum accessibility to SpyTag-fusions. A unique cysteine was introduced in SpyCatcher2.1 E77A at three positions. Cysteine introduction sites were selected for efficient coupling to iodoacetyl-activated (SulfoLink) beads as well as for minimal disruption to SpyTag/SpyCatcher2.1 binding, by having the cysteine substitutions distal from the CnaB2 reactive triad (Fig.聽2c). Three mutations were examined: N-terminal cysteine (N-term Cys) preceding the coding sequence of SpyCatcher2.1, S49C and S55C34,35 (Fig.聽2c). After immobilizing SpyCatcher2.1 E77A with each cysteine mutation, purified SpyTag-MBP was mixed with the resin, washed, and eluted. All Cys mutants showed similar SpyTag-MBP retention and minimal leak-through during washes, but S49C had the least SpyTag-MBP left on the resin after 4 elutions (Fig.聽2d and Supplementary Fig.聽1). Therefore, the finalized protein for Spy Go purification was SpyCatcher2.1 S49C E77A, hereafter termed SpyDock (amino acid sequence in Supplementary Fig.聽2). SpyDock can be coupled to SulfoLink resin to achieve 14.2鈥塵g of SpyDock protein per mL of resin (Supplementary Fig.聽3).To test how well SpyDock bound to SpyTagged proteins, we used isothermal titration calorimetry (ITC) to measure the dissociation constant (Kd) for SpyDock鈥檚 interaction with SpyTag-MBP (Fig.聽3a) or SpyTag002-MBP (Fig.聽3b). Both proteins bound with 1:1 stoichiometry. SpyTag002-MBP (Kd鈥?鈥?.073鈥壩糓) bound ~10-fold tighter than SpyTag-MBP (Kd鈥?鈥?.75鈥壩糓), consistent with the improved properties of the second-generation Tag/Catcher technology23.Fig. 3Affinity of SpyDock for its targets. ITC binding isotherms for SpyDock binding at 25鈥壜癈 to a SpyTag-MBP or b SpyTag002-MBP. Error bars represent the uncertainty in fit to the binding curve using a 1:1 binding modelFull size imagePurification of model proteinsWith the Spy Go resin finalized, we sought to evaluate the capability of the system for purifying proteins from cellular material. Affinity purification becomes easier when proteins are highly over-expressed, so we challenged the system by doping a low amount of purified SpyTag-MBP into clarified E. coli lysate at 0.36鈥塵g protein per g of wet cell weight. As a comparison, we typically obtain ~5鈥塵g of purified recombinant protein per g of E. coli wet cell weight26. A series of optimizations established that 2.5鈥塎 imidazole at neutral pH was efficient at eluting SpyTag from SpyDock, with lower imidazole concentrations used in the wash buffer36. Imidazole is an inexpensive and highly soluble reagent, already present in most biochemistry laboratories. As a bench mark for Spy Go purification, the SpyTag protein also had a His6-tag, so that the same protein and lysate could be compared for the common approach of Ni-NTA purification. The same amount of His-tagged SpyTag-MBP in clarified E. coli lysate was mixed with Ni-NTA resin at the same volume. Spy Go-based purification enabled purification of SpyTag-MBP with higher purity (98.9鈥壜扁€?.5%) than via Ni-NTA purification (66.4鈥壜扁€?.9%), as seen in the pooled elution fractions measured by gel densitometry (Fig.聽4a, b). The ability of the resin to bind and sequester a low concentration of input protein also indicates the sensitivity of Spy Go as a purification platform.Fig. 4Spy Go from bacterial expression. a SpyTag-MBP was purified from E. coli clarified lysate by Spy Go. Protein: input SpyTag-MBP protein. T: total pooled elutions. Purity of T was determined by densitometry (right); gray represents background lane intensity (mean鈥壜扁€?鈥塻.d., n鈥?鈥?). b Ni-NTA purification of SpyTag-MBP from the same lysate via its His-tag. c Spy Go purification of scPvuII-SpyTag (SpyTag at an internal loop, shown schematically) from bacterial lysate. d Spy Go purification of the nanobody 伪DR5-SpyTag (C-terminal fusion) from bacterial lysate. All fractions were analyzed by SDS-PAGE with Coomassie stainingFull size imageSpyTag is the most widely used partner of SpyCatcher in the literature22,37,38, so our purification approaches focused on this tag version. However, we also validated that Spy Go was efficient for purification of a SpyTag002-fusion23 (Supplementary Fig.聽4A).Additionally, Spy Go resin was still capable of purifying SpyTag-MBP from bacterial lysate after storage of resin in 20% (v/v) ethanol for 12 weeks. Therefore Spy Go resin showed good stability, as long as microbial growth was inhibited (Supplementary Fig.聽4B).Spy Go allowed purification with SpyTag at different sitesSome affinity tags can only be placed at either the N- or C-terminus, thus restricting experimental flexibility, especially if a functional site of the protein of interest is close to a terminus1,8,39. To test capture of SpyTag inserted in the loop of a protein, we generated a single-chain dimer of the restriction enzyme PvuII. scPvuII-SpyTag was purified efficiently from soluble E. coli expression using Spy Go (Fig.聽4c). To test capture of SpyTag at the C-terminus of a protein, we expressed a nanobody to Death Receptor 5 (DR5) (伪DR5-SpyTag) in the cytosol of E. coli and showed efficient purification using Spy Go (Fig.聽4d). A single round of purification is not expected to achieve 100% purity and proteins may be subsequently polished using standard methods such as size-exclusion chromatography or ion-exchange chromatography, as may assist subsequent applications.To test purification of a different class of protein, the enhanced green fluorescent protein mClover3 was genetically fused with SpyTag at its N-terminus and expressed solubly in E. coli. After purification and dialysis, the fluorescence of mClover3 was comparable to the same protein purified via its His-tag using Ni-NTA (Supplementary Fig.聽5), supporting that Spy Go purification maintained the functionality of purified proteins. From lysate purifications, the resin capacity ranged from 4鈥?3鈥塵g of protein per mL of resin, depending on the location of the SpyTag and protein used.We established effective regeneration of Spy Go resin via stripping using 4鈥塎 imidazole, guanidinium hydrochloride, and NaOH (Supplementary Fig.聽6A). Following regeneration of the resin, the smaller protein 伪DR5-SpyTag was purified by Spy Go, while SpyTag-MBP (previously purified using the same resin batch) was not detected (Supplementary Fig.聽6B). Purification of 伪DR5-SpyTag using new resin or regenerated resin gave similar results (Supplementary Fig.聽6B).Spy Go allowed purification from mammalian expressionSome affinity tags do not work well in particular cell expression systems, requiring extra materials or steps40. Transient mammalian expression is becoming a dominant route for the production of many therapeutic and research proteins because of the rapid pipeline, high degree of folding quality control, and the native post-translational modifications (notably N-linked glycosylation)41. Epithelial cell adhesion molecule (EpCAM) is a widely used marker for capture of circulating tumor cells and its adhesive interactions may affect metastasis42,43. We cloned the soluble extracellular region of EpCAM with SpyTag and His-tag at the C-terminus and expressed this glycoprotein through transient transfection in HEK293T cells. After expression, the same volume of supernatant from the cell culture was incubated with Spy Go resin or Ni-NTA resin. EpCAM-SpyTag was efficiently purified using Spy Go, as with Ni-NTA (Fig.聽5a, b). The heterogeneous gel mobility of EpCAM-SpyTag is expected because of various glycoforms being secreted.Fig. 5Spy Go from mammalian expression. a HEK293T cells were transfected with the extracellular region of EpCAM fused to SpyTag and a His-tag (EpCAM-SpyTag). EpCAM-SpyTag was purified from the clarified cell supernatant using Spy Go. Fractions were analyzed by SDS-PAGE with Coomassie staining. T: total pooled elutions. Resin: resin post-elution. b Ni-NTA purification of EpCAM-SpyTag as in a. c Spy Go purification of CyRPA-SpyTag from Expi293HEK cells as in aFull size imageWe previously expressed constructs with a His-tag, to allow bench-marking of our purification, but we also purified Cysteine-rich Protective Antigen (CyRPA) with a SpyTag but no His-tag. CyRPA from Plasmodium falciparum is a promising blood-stage antigen for malaria vaccination44. CyRPA-SpyTag was transiently expressed in Expi293HEK cells and efficiently purified by Spy Go (Fig.聽5c). Overall, we have shown that Spy Go is a viable platform for the purification of proteins with SpyTag at the N-terminus, C-terminus or an internal site from either bacterial or mammalian expression systems.Oligomeric assembly of SpyTag-fusionsAn important feature of using SpyTag as the peptide tag for a protein of interest is that SpyTag can then enable a range of subsequent bioconjugation reactions. For example, SpyTag allows irreversible immobilization on surfaces or hydrogels29,30,45, or high-level multimerization on virus-like particles to accelerate vaccine generation24,26,37. To extend the application of SpyTag-fusions, here we also explored the ability for rapid assembly of dimers, trimers, tetramers etc. of the protein of interest. We aimed to prepare a panel of parallel coiled coils fused to SpyCatcher002 that could spontaneously self-assemble into different homo-oligomers with valency from 2鈥? (Fig.聽6a). oDi would represent a dimeric oligomer and oTri a trimeric oligomer and so on.Fig. 6Construction of oligomeric toolbox. a Spy Oligomerization Toolbox from establishing a panel of coiled coils with valency from 2 to 7 for fusion to SpyCatcher002. The coiled coils are presented with each color representing one chain and the N-terminus colored as a blue ball. The C-terminus of SpyCatcher002, where it will be linked to the coiled coil, is colored as a red ball. b DLS to show assembly of each SpyCatcher002-oligomer, with hydrodynamic radius (Rh) (mean鈥壜扁€?鈥塻.d., n鈥?鈥?0) for each assembly. c SEC-MALS to show assembly of each SpyCatcher002-oligomer. The peak shows the normalized absorbance units (AU) at 280鈥塶m of the SpyCatcher002-oligomer species from SEC. The horizontal line shows the distribution of molar mass (g/mol) of the species in the peak from MALS. Expected and observed molecular weight (Mr) is shown alongside, with error bars representing the uncertainty in fit to the molar mass curveFull size imageFor oDi, oTri and oHex, we selected coiled coils that were previously computationally designed by the Woolfson laboratory, based on knowledge of key side-chain interactions determining valency and stability, and validated after solid-phase peptide synthesis46,47. We increased the repeat number to favor high stability48, genetically fused the coiled coils to SpyCatcher002, and expressed constructs in the cytosol of E. coli. The best oTet was the coiled coil from VASP (vasodilator-stimulated phosphoprotein)49. For oPent, we selected the coiled coil from COMP (cartilage oligomeric matrix protein)50. Finally for oHept, we tested the coiled coil from IMX313, originally adapted from complement C4 binding protein51. These platforms, containing a His-tag, were purified in good yield after Ni-NTA and gel-filtration chromatography. We validated the composition of each of the coiled coil subunits by electrospray ionization mass spectrometry (Supplementary Fig.聽7).The purified SpyCatcher002-oligomers were subjected to dynamic light scattering (DLS) to verify monodispersity and correct assembly. DLS indicated increasing valency from oDi to oHept based on the increasing hydrodynamic radius (Rh) (Fig.聽6b). We further analyzed the oligomers by size-exclusion chromatography/multi-angle light scattering (SEC-MALS). SEC-MALS also analyzes uniformity but additionally characterizes the molecular weight of the assembly. The observed molecular weights were within 10% of the expected assembled molecular weights (Fig.聽6c), suggesting successful assembly by each oligomer from oDi up to oHept.In our hands, the previously designed tetrameric coiled coil, CC-Tet46, when fused to SpyCatcher002, had an observed mass that deviated substantially from the expected mass (Supplementary Fig.聽8). We also found that SpyCatcher002 fused to the designed CC-Hept47 gave satisfactory assembly (Supplementary Fig.聽8), but expressed poorly and was prone to aggregate. Therefore the final Spy oligomerization toolbox was a mixture of computationally designed or natural coiled coils (amino acid sequences shown in Supplementary Fig.聽9).Oligomeric assembly of a nanobodyDR5 is a pro-apoptotic receptor overexpressed on many cancer cells. DR5 activation promotes cell death in response to binding of TNF-related apoptosis-inducing ligand (TRAIL)52. DR5 targeting shows promise as a target for cancer therapy53. There has been extensive interest in the clustering of DR553,54,55,56,57,58,59,60, because dimeric anti-DR5 IgG typically causes weak activation of DR5 signaling53. Previous attempts at clustering focused on linear chains of anti-DR5 agonists. We explored the use of multivalent display of an agonist nanobody (伪DR5), to evaluate the effect of increasing nanobody valency on triggering cancer cell death. Nanobodies are a convenient monomeric scaffold, with the single chain easily expressed in E. coli. 伪DR5 was fused genetically to SpyTag and was purified efficiently via Spy Go (Fig.聽4d). We then mixed this nanobody with the Spy-coiled coil platforms for convenient modular preparation of dimers, trimers etc. up to heptamers.伪DR5-SpyTag was incubated with each SpyCatcher002-oligomer and efficient covalent reaction was confirmed by SDS-PAGE. Spy Go also enabled us to remove excess 伪DR5-SpyTag from the mixture, by incubating with Spy Go resin to recapture unconjugated 伪DR5-SpyTag, leaving only oligomer:nanobody complex in the flow-through (Fig.聽7a, b).Fig. 7Oligomer panel tested for cancer cell killing. a Cartoon depicting depletion of free SpyTag-ligand using Spy Go resin. b Coupling of SpyTag-fusion to coiled coil series. 伪DR5-SpyTag was incubated with each SpyCatcher002-coiled coil and analyzed by SDS-PAGE with Coomassie staining. In Recaptured lanes, excess 伪DR5-SpyTag was removed from the coiled coil conjugate by an additional passage through Spy Go resin. c Dose-response curve of MDA-MB-231 cell viability when treated with different concentrations of 伪DR5 conjugated to each SpyCatcher002-oligomer platforms. The line at 50% cell viability shows the cut-off for EC50 calculation. The x-axis is normalized to the concentration of 伪DR5 monomer. Error bars represent mean 卤1鈥塻.d., n鈥?鈥?. d MDA-MB-231 viability upon incubation as in c with the building blocks of 伪DR5 alone or coiled coils alone. Error bars represent mean鈥壜扁€?鈥塻.d., n鈥?鈥?Full size imageDR5 signal activation by the nanobody oligomeric seriesTo assess the potency of these oligomeric SpyCatcher002 platforms, we chose a TRAIL-sensitive human breast cancer cell-line MDA-MB-23154. We tested a broad range of doses, from 0.1鈥塸M鈥?00鈥塶M for each SpyCatcher002-oligomer:伪DR5-SpyTag and compared death induction after 24鈥塰 of treatment. While monomeric unconjugated 伪DR5-SpyTag, dimeric, trimeric, and tetrameric conjugates failed to display any cell killing within this range, we observed a clear stoichiometry-dependent dose-response for higher-order 伪DR5-conjugates (Fig.聽7c). All higher-order conjugates (pentameric, hexameric and heptameric) showed potency at sub-nanomolar concentrations. The pentameric complex resulted in killing of 50% of the cells (EC50) at 0.9鈥塶M 伪DR5. Similarly, the hexamer gave a EC50 of 0.42鈥塶M whereas the heptamer had high potency at 0.31鈥塶M EC50 (Fig.聽7c). As a control, we found that unconjugated SpyCatcher002-oligomer constructs did not elicit any dose-dependent loss of cell viability (Fig.聽7d).DiscussionWe have established a system for purification of SpyTag-fused proteins using an immobilized unreactive SpyCatcher variant. The engineering of SpyDock enables efficient binding of SpyTag, facilitating capture and elution of the protein of interest for the different SpyTag iterations. His-tag/Ni-NTA purification has been optimized over decades but our initial results suggest favorable comparison to Spy Go for purification from bacteria. We also demonstrated that mammalian proteins can be efficiently purified using SpyTag. As anticipated from the flexible location of SpyTag for covalent reaction with SpyCatcher21,37,38,45,61, SpyTag can be located at either terminus of the protein of interest or at an internal site for Spy Go purification. We have also shown that Spy Go resin can be effectively regenerated and retains its function after storage for months. The current elution conditions for Spy Go are unconventional (2.5鈥塎 imidazole in Tris-phosphate buffer). Nevertheless, we have observed little aggregation and good yields for the different classes of proteins so far explored. After dialyzing away imidazole, we validated biological activity for mClover3 fluorescence and DR5 agonist cellular activation. We previously demonstrated the tolerance of various enzymes and fluorescent proteins to similar high imidazole concentrations36.Beyond primary purification, Spy Go also facilitates the generation of purified nanoassembly preparations. We could remove the excess 伪DR5-SpyTag from each conjugated SpyCatcher002-oligomer:伪DR5-SpyTag complex by incubating the mixture with Spy Go resin. Since SpyTag reacts irreversibly with SpyCatcher002, the reconstitution reaction renders the conjugated SpyTag unavailable for binding to SpyDock.Modularity is a defining principle in accelerating biological research, moving from painstaking case-by-case artisanal optimization to an efficient and automatable assembly-line62. Previous coiled coils were already individually explored as genetic fusion partners for proteins25,63,64,65, however, optimizations would be needed for each distinct protein24,37,65. Examples include heterogeneous populations of 5鈥? oligomers instead of a monodisperse heptamer66, failed assembly of a trimer-fusion protein67, and severe degradation of dimers in E. coli cytosol68,69.The characterized Spy-based oligomerization toolbox can alleviate this problem, separating the expression/folding/post-translational modification of coiled coils and cargo protein before uniting via SpyTag/SpyCatcher. Spy Go-purified 伪DR5-SpyTag readily reacted to completion with multivalent SpyCatcher002-oligomer to form a panel of valency-defined platforms to understand the effect of higher valencies on cellular signaling and the downstream apoptotic effect. Up until now, most multimeric anti-DR5 or soluble TRAIL constructs consist of a linear arrangement linked by simple Gly-Ser linkers extending to 5 repeats55, 6 repeats56,57 and in one case 8 repeats57 of the agonist against DR553. However, the formation of a linear complex may not mimic the clustering by the native TRAIL to DR5 receptor. A 鈥渃ombody鈥?comprising a genetic fusion of the coiled coil COMP with an agonistic 伪DR5 was previously made70, but direct fusion may not be optimized for other proteins or coiled coil combinations, and no higher valencies were reported. We note that we have characterized SpyCatcher002-coiled coil valency by DLS and SEC-MALS at micromolar concentrations. Both natural and synthetic multimers may start to dissociate as one approaches low nanomolar or picomolar concentrations, where cells may still respond to DR5 clustering but bulk biophysical assays become very difficult46,47. In future work, single-molecule assays may be the best way to understand molecular and cellular behavior in this low concentration regime71,72.We envision that the oligomerizing SpyCatcher002-coiled coil platforms can advance the study of valency-dependence on diverse cellular signaling processes73,74. By combining Spy Go purification with coiled coil nanoassembly, SpyTagging may help to accelerate the exploration and exploitation of protein space.MethodsPlasmids and cloningConstructs were cloned by standard PCR methods and assembled using Gibson assembly. Inserts were verified by Sanger sequencing. pDEST14-SpyCatcher2.1 was derived from pDEST14-SpyCatcher00223 (GenBank MF974388 and Addgene plasmid ID 102827) with additional A89P, Q97D and K108E mutations (see below). pDEST14-SpyCatcher2.1 S49C E77A (SpyDock) (Supplementary Fig.聽2, GenBank MK637462, Addgene plasmid ID 124618) has the organization: His6, SpyCatcher2.1 with E77A S49C mutations, GSSGS. pDEST14-SpyCatcher2.1 E77X S49C has the same organization as SpyDock except with E77X mutations instead, with X being D, G, N, Q, S, T, or V. pDEST14-SpyCatcher2.1 E77A N-term Cys has the same organization as SpyDock except with a cysteine-anchoring mutation at the 6th amino acid residue preceding SpyCatcher2.1 E77A instead of S49C. pDEST14-SpyCatcher2.1 S55C E77A has the same organization as SpyDock except with a S55C mutation instead of S49C mutation. pET28a-SpyTag-MBP21 (Addgene plasmid ID 35050) and pET28a-SpyTag002-MBP23 (GenBank MF974389 and Addgene plasmid ID 102831) were as described. pENTR4-EpCAM-SpyTag (GenBank MK637463) was derived from pENTR4-EpCAM-SnoopTagJr30 (GenBank MH511516) with the organization: tissue plasminogen activator (tPA) secretion leader sequence, extracellular domain of human EpCAM protein (EpCAM; residue 24鈥?65), (GSG)2, SpyTag, GEGS, His6. pENTR4-LPTOS-CyRPA-SpyTag26 (GenBank MH425516) was previously published. pET28a-伪DR5-SpyTag (GenBank MK637464) was derived from pET28a-SnoopTag-伪DR5-SpyTag (GenBank KU500643)54 with the organization: 伪DR5 (4E6 nanobody55), (GGGGS)2, SpyTag. pET28a-SpyTag-mClover3 has the organization: SpyTag, SGGGSG, mClover375, GSGSGS, His6. pET28a-scPvuII-SpyTag (GenBank MK637465) has the organization: His6, SSG, PvuII from Proteus vulgaris, GSG, TEV cleavage site, GGSG, SpyTag, GSGG, PvuII. SpyTag along with surrounding spacers was inserted into a loop of the first PvuII between amino acid residues 75 and 76. PvuII constructs had the D58A mutation to block DNA cleavage. pDEST14-SpyCatcher002 was linked to coiled coil inserts consisting of oDi46 (GenBank MK637466, Addgene plasmid ID 124661), oTri46 (GenBank MK637467, Addgene plasmid ID 124662), oTet49 (GenBank MK637468, Addgene plasmid ID 124663), oPent50 (GenBank MK637469, Addgene plasmid ID 124664), oHex47 (GenBank MK637470, Addgene plasmid ID 124670), oHept51 (GenBank MK637471, Addgene plasmid ID 124671), CC-Tet46 (GenBank MK637472) or CC-Hept47 (GenBank MK637473). The constructs have the organization: His6, DYDIPTT spacer, TEV cleavage site, SpyCatcher002, GSSGSGSGS, coiled coil inserts, GSGSG, C-tag. Coiled coil DNA was synthesized by IDT DNA Technologies (Supplementary Fig.聽9). oDi, oTri, oHex, CC-Tet, and CC-Hept have 5 heptad repeats, instead of 4 repeats reported in the literature, to increase the stability of coiled coil assembly48.SpyDock rational designTo complement the increase in positively charged residues of SpyTag002 compared to SpyTag, mutations Q97D and K108E were made on SpyCatcher002 to increase the negatively charged residues, making the SpyDock precursor, SpyCatcher2.1, to improve the electrostatic interaction with the positively-charged SpyTag or SpyTag002 (Fig.聽2c). Residues Y83 and E85 within the long A79-A89 loop in SpyCatcher make key interactions with residues Y9 and K10 of SpyTag in the SpyTag/SpyCatcher crystal structure76. These wild-type residues were also positively selected during directed evolution to produce SpyTag002, supporting the importance of the residues for rapid isopeptide bond formation23. Inclusion of prolines in turns and loops has previously been shown to stabilize proteins36,77,78,79. Hence, the A89P mutation was included to stabilize the A79-A89 loop (Fig.聽2c). The E77 was targeted to nullify the isopeptide bond formation, with A, D, G, N, Q, S, T, and V mutations as candidates (Fig.聽1). These mutations were postulated to enable better binding and retention of SpyTag to SpyDock during the binding and washing steps, along with easier elution.Bacterial protein expressionpET28a-SpyTag-MBP, pET28a-SpyTag002-MBP, and pET28a-SpyTag-mClover3 were transformed into chemically competent E. coli BL21 (DE3) RIPL (Agilent Technologies). pET28a-scPvuII-SpyTag was transformed into T7 Express lysY/Iq (NEB). pDEST14-SpyCatcher2.1 S49C E77A (SpyDock), pDEST14-SpyCatcher2.1 S49C E77X variants, pDEST14-SpyCatcher2.1 E77A N-term Cys, pDEST14-SpyCatcher2.1 S55C E77A and pDEST14-SpyCatcher002-coiled coil fusions were transformed into chemically-competent E. coli C41 (DE3), a kind gift from Anthony Watts (University of Oxford). pET28a-伪DR5-SpyTag was transformed into E. coli BL21 (DE3) RIPL containing a gene encoding phosphogluconolactonase, to degrade 6-phosphogluconolactone, which promotes protein gluconoylation80. The cells were plated on LB agar supplemented with 50鈥壜礸/mL kanamycin (pET28a) or 100鈥壜礸/mL ampicillin (pDEST14). For 伪DR5-SpyTag, 34鈥壜礸/mL chloramphenicol was added alongside kanamycin throughout. The plates were incubated at 37鈥壜癈 overnight until colonies were observed.Single colonies of pET28a-SpyTag-MBP, pET28a-SpyTag002-MBP, pET28a-SpyTag-mClover3, pET28a-scPvuII-SpyTag, and pDEST14-SpyDock, all variants of pDEST14-SpyCatcher2.1 S49C E77X, pDEST14-SpyCatcher2.1 E77A N-term Cys, pDEST14-SpyCatcher2.1 E77A S55C, and all variants of pDEST14-SpyCatcher002-coiled coils were picked and inoculated into 10鈥塵L LB medium supplemented with 50鈥壜礸/mL kanamycin (pET28a) or 100鈥壜礸/mL ampicillin (pDEST14), incubated at 37鈥壜癈 with shaking at 200鈥塺pm for 16鈥塰. The cultures were then inoculated into 1鈥塋 LB supplemented with 50鈥壜礸/mL kanamycin (pET28a) or 100鈥壜礸/mL ampicillin (pDEST14) and 0.8% (w/v) glucose (except for pDEST14-SpyCatcher002-coiled coils), incubated at 37鈥壜癈 with shaking at 200鈥塺pm until A600 0.5鈥?.6, when the cultures were induced with 0.42鈥塵M isopropyl 尾-d-1-thiogalactopyranoside (IPTG) (Fluorochem). Cultures of pET28a-SpyTag-MBP, pET28a-SpyTag002-MBP, pET28a-SpyTag-mClover3, pET28a-scPvuII-SpyTag, and pDEST14-SpyDock, all variants of pDEST14-SpyCatcher2.1 S49C E77X, pDEST14-SpyCatcher2.1 E77A N-term Cys, and pDEST14-SpyCatcher2.1 E77A S55C were grown further for 4鈥塰 with shaking at 200鈥塺pm at 30鈥壜癈. Cultures of pDEST14-SpyCatcher002-oDi, pDEST14-SpyCatcher002-oTri, pDEST14-SpyCatcher002-oTet, pDEST14-SpyCatcher002-oPent, and pDEST14-SpyCatcher002-CC-Tet were grown further for 16鈥塰 with shaking at 200鈥塺pm at 22鈥壜癈. Cultures of pDEST14-SpyCatcher002-oHex, pDEST14-SpyCatcher002-oHept and pDEST14-SpyCatcher002-CC-Hept were grown for 4鈥塰 with shaking at 200鈥塺pm at 37鈥壜癈.A single colony of pET28a-伪DR5-SpyTag was picked and inoculated into 1鈥塋 auto-induction medium (AIMLB0205 from Formedium) supplemented with 50鈥壜礸/mL kanamycin and 34鈥壜礸/mL chloramphenicol, incubated at 30鈥壜癈 with shaking at 200鈥塺pm for 24鈥塰. All E. coli-expressed proteins were harvested by centrifugation at 4000脳g for 15鈥塵in at 4鈥壜癈 prior to purification.Mammalian protein expressionEpCAM-SpyTag was expressed in adherent HEK293T cells. HEK293T cells were cultured in T175 adhesive culture flasks (Corning) with Dulbecco鈥檚 Modified Eagle鈥檚 Medium (DMEM) (Sigma-Aldrich) high glucose with 10% (v/v) Fetal Bovine Serum (Sigma-Aldrich), 2鈥塵M l-glutamine, 100鈥塙/mL penicillin, and 100鈥壩糶/mL streptomycin (Thermo Fisher Scientific) at 37鈥壜癈 with 5% CO2. Before transfection, the cells were seeded into a T875 5-layer flask (Corning) and upon reaching 50% confluency, they were transferred into serum-free media (DMEM, 2鈥塵M glutamine, 50鈥塙/mL penicillin, 25鈥塵M HEPES added) and mixed with 30鈥壩糶 pENTR4-EpCAM-SpyTag plasmid per 7.5鈥塵L of media for each flask layer. After 15鈥塵in, 2.5鈥塵L media containing 36鈥壩糶/mL polyethyleneimine (Sigma-Aldrich) was added to each layer. 10鈥塵L media containing 4.4鈥壩糓 valproic acid (Sigma-Aldrich), 100鈥塙/mL penicillin, and 100鈥壩糶/mL streptomycin was added to each layer 16鈥?0鈥塰 later. Cells were then incubated at 37鈥壜癈 with 5% CO2 for another 6 days.CyRPA-SpyTag was expressed in suspension Expi293HEK cells (Thermo Fisher Scientific). Expi293HEK cells were cultured in Expi293 expression media (Thermo Fisher Scientific) with 50鈥塙/mL penicillin/streptomycin at 37鈥壜癈 with 7% CO2 shaking at 110鈥?25鈥塺pm. Transient transfection of pENTR4-LPTOS-CyRPA-SpyTag was done using the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific). Cells at a density of 2.5鈥壝椻€?06 cells/mL were transfected with 2.7鈥壩糒 ExpiFectamine 293 Reagent per 1鈥壩糶 of pENTR4-LPTOS-CyRPA-SpyTag plasmid. After 16鈥?8鈥塰, ExpiFectamine transfection enhancers (Thermo Fisher Scientific) were added and the cell supernatant was harvested 4 days post-transfection.The cell supernatants were harvested by addition of cOmplete鈩? Mini, EDTA-free Protease Inhibitor Cocktail (Roche), centrifuged at 1000脳g for 3鈥塵in, and filtered through a 0.45 渭m syringe filter to remove cell debris. 2.5% of 10脳Ni-NTA or 10脳TP buffer (250鈥塵M orthophosphoric acid adjusted to pH 7.0 with Tris base) was added for pH adjustment before affinity chromatography purification.Protein purification by Ni-NTAPurifications were done at 4鈥壜癈 throughout. All E. coli-grown constructs (except 伪DR5-SpyTag, scPvuII-SpyTag, SpyTag-mClover3, SpyCatcher002-oHex, SpyCatcher002-oHept and SpyCatcher002-CC-Hept) were resuspended in 1脳Ni-NTA buffer (50鈥塵M Tris-HCl, 300鈥塵M NaCl pH 7.8; SpyDock along with all variants of SpyCatcher2.1 S49C E77X, SpyCatcher2.1 E77A N-term Cys, SpyCatcher2.1 E77A S55C and SpyCatcher002-oPent had additional 10鈥塵M 2-mercaptoethanol) with cOmplete鈩? Mini, EDTA-free Protease Inhibitor Cocktail and 1鈥塵M phenylmethylsulfonyl fluoride (PMSF). Cells were lysed by addition of 100鈥壩糶/mL lysozyme (Sigma-Aldrich) and 2鈥塙/mL benzonase (Sigma-Aldrich). The lysate was rotated at 25鈥壜癈 for 30鈥塵in and sonicated on ice for 4鈥壝椻€?鈥塵in with 1鈥塵in rest period at 50% duty cycle. Clarified cell lysates were centrifuged at 30,000鈥?i>g for 30鈥塵in before incubation with Ni-NTA beads (Qiagen) on a rotary shaker for 1鈥塰. The lysate-bead mixture was added onto a polyprep gravity column and washed with 20 packed resin volumes of Ni-NTA wash buffer (10鈥塵M imidazole in Ni-NTA buffer, pH 7.8; SpyDock, all variants of SpyCatcher2.1 S49C E77X, SpyCatcher2.1 E77A N-term Cys, SpyCatcher2.1 E77A S55C and SpyCatcher002-oPent had 10鈥塵M 2-mercaptoethanol for the first 10 packed resin volumes; SpyCatcher002 coiled coil fusions used 75鈥塵M imidazole in Ni-NTA buffer) and eluted with Ni-NTA elution buffer (200鈥塵M imidazole in Ni-NTA buffer, pH 7.8; SpyCatcher002 coiled coil fusions used 350鈥塵M imidazole in Ni-NTA buffer) at 4鈥壜癈. Elutions were monitored by A280 and stopped once A280 was 1.0. Proteins were dialyzed against 20鈥塵M Tris-HCl pH 8.0 and concentrated, if necessary, using Vivaspin centrifugal concentrator 5鈥塳Da cutoff (GE Healthcare).SpyDock was further purified on a HiTrap Q HP anion exchange chromatography column (GE Healthcare) connected to an 脛KTA Pure 25 (GE Healthcare) fast protein liquid chromatography (FPLC) system at 4鈥壜癈. The protein was eluted with a linear gradient of 0.2鈥?.35鈥塎 NaCl (in 10鈥塵M Tris-HCl pH 8.0 with 1鈥塵M dithiothreitol) at a flow rate of 2鈥塵L/min at 4鈥壜癈. Peak fractions were verified by SDS-PAGE gels, dialyzed against 20鈥塵M Tris-HCl pH 8.0, and concentrated using Vivaspin centrifugal concentrator 5鈥塳Da cutoff. Typical yield for SpyDock after Ni-NTA and ionic exchange chromatography is approximately 28鈥塵g/L culture.All SpyCatcher002 coiled coil fusions were further purified by gel filtration chromatography on a pre-equilibrated HiLoad 16/600 Superdex 200鈥塸g column connected to 脛KTA Pure 25 FPLC system. The mobile phase was 50鈥塵M Tris-HCl, 150鈥塵M NaCl pH 8.0 at a flow-rate of 1鈥塵L/min at 4鈥壜癈 with A280 monitored throughout. Fractions were collected corresponding to the size of the oligomeric SpyCatcher002 coiled coil complex: SpyCatcher002-oDi (75鈥?8鈥塵L), SpyCatcher002-oTri (69鈥?2鈥塵L), SpyCatcher002-oTet (66鈥?0鈥塵L), SpyCatcher002-oPent (62鈥?5鈥塵L), SpyCatcher002-oHex (61鈥?5鈥塵L), SpyCatcher002-oHept (56鈥?0鈥塵L). All SpyCatcher002-coiled coil fusions had a yield of approximately 3鈥塵g/L culture, following Ni-NTA and gel filtration chromatography.Protein purification by refoldingSpyCatcher002-oHex, SpyCatcher002-oHept, and SpyCatcher002-CC-Hept were found in inclusion bodies and so were purified by refolding. Cells were resuspended in 100鈥塵M Tris-HCl pH 8.0 with cOmplete鈩? Mini, EDTA-free Protease Inhibitor Cocktail and 1鈥塵M PMSF. Cells were lysed by addition of 100鈥壩糶/mL lysozyme and 2鈥塙/mL benzonase and rotated at 25鈥壜癈 for 30鈥塵in. There was one freeze-thaw cycle from 鈭?0鈥壜癈 to 25鈥壜癈, followed by addition of 0.5% (v/v) Triton X-100 and sonication on ice for 4鈥壝椻€?鈥塵in with 1鈥塵in rest period at 50% duty cycle. Clarified cell lysates were centrifuged at 30,000脳g for 30鈥塵in. The cell pellet was washed with 2鈥壝椻€塒BS鈥?鈥?.1% (v/v) Triton X-100 and then 2鈥壝椻€塒BS, with centrifugation at 25,000脳g for 20鈥塵in between steps. The inclusion body pellet was resuspended in 8鈥塎 urea in 50鈥塵M Tris-HCl pH 8.0 and refolded by diluting 50-fold into refolding buffer of 0.1鈥塎 Tris-HCl pH 8.0, 0.4鈥塎 l-arginine and 0.1鈥塎 PMSF (for SpyCatcher002-oHept, 5鈥塵M reduced glutathione and 0.5鈥塵M oxidized glutathione were present) for 40鈥塰. The mixture was filtered through a 0.45-渭m membrane before incubation with Ni-NTA beads on a rotary shaker for 1鈥塰. The lysate-bead mixture was added onto a polyprep gravity column and washed with 20 resin volumes of Ni-NTA wash buffer (with 75鈥塵M imidazole; SpyCatcher002-oHept had 10鈥塵M 2-mercaptoethanol for the first 10 packed resin volumes) and eluted with Ni-NTA elution buffer (with 350鈥塵M imidazole) at 4鈥壜癈. Elutions were monitored by A280 and stopped once A280 was 1.0. Proteins were dialyzed against 20鈥塵M Tris-HCl pH 8.0 and concentrated to suitable working concentrations, if needed, using Vivaspin centrifugal concentrator 5鈥塳Da cutoff (GE Healthcare).SpyCatcher2.1 E77A variants coupling to SulfoLink resinPurified SpyCatcher2.1 E77A with various cysteine anchoring residues (N-term Cys, S55C, and S49C) were coupled to SulfoLink Coupling Resin (Thermo Fisher Scientific) according to the manufacturer鈥檚 protocol at 25鈥壜癈. In short, 20鈥塵g of protein for every 1鈥塵L of resin was reduced by 1鈥塵M tris(2-carboxyethyl)phosphine (TCEP) (Fluorochem, UK) for 30鈥塵in, prior to mixing end-over-end with equilibrated resin for 15鈥塵in and leaving to stand for 30鈥塵in covered by foil. Protein flow-through was aspirated and resin was washed with 10 resin volumes of coupling buffer (50鈥塵M Tris-HCl, 5鈥塵M EDTA, pH 8.5). The resin was blocked with 50 mM L-cysteine-HCl in coupling buffer (MP Biomedicals), mixed end-over-end for 15鈥塵in, and left to stand for 30鈥塵in. The resin was then washed with 10 resin volumes of 1鈥塎 NaCl, and stored in 1xTris-phosphate (TP) buffer (25鈥塵M orthophosphoric acid adjusted to pH 7.0 with Tris base), before adding 0.05% (w/v) NaN3 and storing at 4鈥壜癈.Isothermal titration calorimetrySpyDock was trapped as a monomer by reduction with 2.5鈥塵M TCEP in 50鈥塵M Tris-HCl with 5鈥塵M EDTA at pH 8.5 and by cysteine modification with 20鈥塵M iodoacetamide in 50鈥塵M Tris-HCl with 5鈥塵M EDTA at pH 8.5 for 30鈥塵in in the dark to produce carbamidomethylated SpyDock. Excess TCEP and iodoacetamide were removed by dialysis. SpyTag-MBP, SpyTag002-MBP and the modified SpyDock were subsequently dialyzed twice into 20鈥塵M HEPES pH 7.0 with 150鈥塵M NaCl. Experiments were carried out using a Microcal PEAQ-isothermal titration calorimetry (ITC) calorimeter (Malvern) at 25鈥壜癈 in 20鈥塵M HEPES pH 7.0 with 150鈥塵M NaCl. 20鈥壩糓 SpyDock was used in the cell and titrated with 20 injections of 210鈥壩糓 SpyTag-MBP or SpyTag002-MBP in the syringe. Analyses were carried out using a 1:1 binding model with the MicroCal PEAQ-ITC Analysis software version 1.1.0.1262.Purification by Spy GoTo determine the best cysteine anchoring site, 50鈥壩糒 packed resin with SpyCatcher2.1 E77A N-term Cys, S55C, or S49C was mixed with 500鈥壩糒 TP buffer containing 0.09鈥塵g (10脳 lower than SpyCatcher2.1 E77A available on resin) of Ni-NTA-purified SpyTag-MBP in an Eppendorf tube through batch chromatography. The low amount of SpyTag-MBP introduced was to test the sensitivity of Spy Go purification. The protein was mixed with the resin for 1鈥塰 with tumbling at 4鈥壜癈. Standard Spy Go batch chromatography purification is as follows: the resin was washed 4鈥壝椻€墂ith 10 resin volumes of TP buffer, with incubation at 4鈥壜癈 shaking at 1,200鈥塺pm for 3鈥塵in. Resin was then centrifuged at 4000脳g for 3鈥塵in at 4鈥壜癈. The protein was eluted with 4鈥壝椻€?.5 resin volume of elution buffer ETP (2.5鈥塎 imidazole in TP buffer).For purification of SpyTag-MBP and SpyTag002-MBP from lysate, 0.09鈥塵g of the Ni-NTA-purified proteins were added into ~0.25鈥塯 wet cell weight of induced BL21 (DE3) RIPL cleared lysate dissolved in 500鈥壩糒 TP buffer in an Eppendorf tube containing 50鈥壩糒 packed Spy Go resin. The protein-lysate was mixed with the resin for 1鈥塰 tumbling at 4鈥壜癈. Standard Spy Go batch chromatography purification was performed to purify the proteins but washed with wash buffer WTP (500鈥塵M imidazole in TP buffer).EpCAM-SpyTag was purified from the supernatant of HEK293T cells by mixing 50鈥塵L of the supernatant with 0.5鈥塵L packed Spy Go resin. CyRPA-SpyTag was purified from the supernatant of Expi293HEK cells by mixing 3.5鈥塵L of the supernatant with 50鈥壜礚 of packed Spy Go resin. Both were rolling for 1鈥塰 at 4鈥壜癈. The mixture was then purified by standard gravity column chromatography: resin was washed 4脳 with 10 resin volumes of TP buffer and eluted with 6鈥壝椻€? resin volume of ETP buffer (4鈥壝椻€? resin volume for CyRPA-SpyTag). Typical yield for EpCAM-SpyTag after Spy Go purification was approximately 2.9鈥塵g/L culture and for CyRPA-SpyTag was approximately 8.9鈥塵g/L culture.For comparison with Ni-NTA resin, the methods described were performed with equivalent volume of packed Ni-NTA resin with the following changes: the cell pellet was resuspended in 50鈥塵M Tris-HCl, 300鈥塵M NaCl pH 7.8, the wash buffer was Ni-NTA wash buffer (30鈥塵M imidazole for SpyTag-MBP, 10鈥塵M imidazole for EpCAM-SpyTag) and elution was carried out with Ni-NTA elution buffer.For purification of 伪DR5-SpyTag, the cell pellet from 1鈥塋 of culture was centrifuged and resuspended in 10鈥塵L TP buffer with 1鈥塵M dithiothreitol, cOmplete鈩? Mini, EDTA-free Protease Inhibitor Cocktail and 1鈥塵M PMSF. Cells were lysed by addition of 100鈥壩糶/mL lysozyme and 2鈥塙/mL benzonase rotated at 25鈥壜癈 for 30鈥塵in, and subsequently sonicated on ice for 4鈥壝椻€?鈥塵in at 50% duty cycle and spun down at 30,000鈥?i>g to remove cell debris. The cleared lysate was mixed with 1鈥塵L of Spy Go resin rolling for 1鈥塰 at 4鈥壜癈. Standard gravity column chromatography purification was performed to purify 伪DR5-SpyTag with washes using WTP buffer.For purification of scPvuII-SpyTag and SpyTag-mClover3, the cell pellet from 0.25鈥塋 of culture was lysed with 5鈥塵L BugBuster reagent per gram of wet cell weight in the presence of 100鈥壜礸/mL lysozyme and 2鈥塙/mL benzonase. The sample was incubated standing at room temperature for 20鈥塵in. After spinning down at 30,000脳g to remove cell debris, the cleared lysate was mixed with Spy Go resin with rolling for 1鈥塰 at 4鈥壜癈. Standard gravity column chromatography purification was performed using WTP buffer for washes and 4鈥壝椻€? resin volume for elution.SpyTag-protein and SpyCatcher reconstitution reactionTo investigate any formation of covalent bond, 5鈥壩糓 SpyCatcher2.1 S49C with E77X (X being A, D, G, N, Q, S, T, or V) was reacted with 2鈥壝椻€塵olar excess of SpyTag-MBP (10鈥壩糓) at 25鈥壜癈 for 24鈥塰 in TP buffer. Reaction was quenched with SDS-PAGE loading buffer [0.23鈥塎 Tris HCl pH 6.8, 24% (v/v) glycerol, 120鈥壩糓 bromophenol blue, 0.23鈥塎 SDS, 100鈥塵M 2-mercaptoethanol] and heating at 95鈥壜癈 for 5鈥塵in in a Bio-Rad C1000 thermal cycler.To form SpyCatcher002-oligomer:伪DR5-SpyTag, 100鈥壩糓 monomeric concentration of SpyCatcher002-oligomer was mixed with 300鈥壩糓 伪DR5-SpyTag in 200鈥壩糒 total volume overnight at 25鈥壜癈 in 50鈥塵M Tris-HCl, 150鈥塵M NaCl, pH 8.0. Excess 伪DR5-SpyTag was removed by recapturing using Spy Go resin by incubating the reaction mixture with 50鈥壩糒 Spy Go resin for 2鈥塰 at room temperature with mixing end-over-end. The mixture was then loaded onto a Micro Bio-Spin column (Bio-Rad) and the conjugated SpyCatcher002-oligomer:伪DR5-SpyTag was recovered by centrifugation at 300鈥?i>g for 1鈥塵in at 4鈥壜癈 into a microcentrifuge tube. This process was repeated once. The protein concentrations were measured by Proteoquant BCA assay (Expedeon).SDS-PAGE and protein purity quantificationSDS-PAGE was performed using 16% Tris-glycine gels in an XCell SureLock system (Thermo Fisher Scientific). Samples were loaded with final concentration of 1x SDS-PAGE loading buffer. For reduced samples, 100鈥塵M 2-mercaptoethanol was added. SDS-PAGE gels were run at 190鈥塚 in 25鈥塵M Tris-HCl, 192鈥塵M glycine, 0.1% (w/v) SDS, pH 8.5. Gels were stained with InstantBlue Coomassie stain (Expedeon), destained with MilliQ water, and imaged using ChemiDoc XRS imager and analyzed with ImageLab (version 6.0.1) (Bio-Rad). In ImageLab, low sensitivity band detection in the final eluted lane (T) was calculated and compared with the protein control lane (Protein) at background subtraction of disk size 2鈥塵m. Percentage purity is defined as 100鈥壝椻€塠target protein Band % in lane T/target protein Band % in lane Protein].Regeneration of Spy Go resinLysate from bacterial expression of SpyTag-MBP was purified using 0.5鈥塵L Spy Go resin as above. The resin was equilibrated with 2鈥壝椻€?0 packed resin volumes of TP buffer before regeneration. Spy Go resin was then regenerated at 25鈥壜癈 by incubating with 3鈥壝椻€?0 packed resin volumes of 4鈥塎 imidazole in TP buffer pH 7.0 for 5鈥塵in each time, washing with 1鈥壝椻€塗P buffer, incubating with 3鈥壝椻€?0 packed resin volumes of 6鈥塎 guanidine hydrochloride pH 2.0 for 5鈥塵in each time, washing with 1鈥壝椻€塗P buffer, incubating with 3鈥壝椻€?0 packed resin volumes of 0.1鈥塎 NaOH for 1鈥塵in each time, and washing with 2鈥壝椻€塗P buffer before storage in 20% (v/v) ethanol in MilliQ water at 4鈥壜癈. Supernatant from each stage was analyzed by SDS-PAGE with Coomassie staining. New resin, resin pre-regeneration and regenerated resin were also analyzed by boiling in SDS-PAGE loading buffer, followed by SDS-PAGE with Coomassie staining. Lysate from bacterial expression of 伪DR5-SpyTag was added to new resin or regenerated resin and purified using the standard Spy Go protocol, before analysis by SDS-PAGE with Coomassie staining. To test capture after storage, Spy Go resin was stored in 20% (v/v) ethanol for 12 weeks at at 4鈥壜癈, before purification of SpyTag-MBP from bacterial lysate following the standard protocol.Mass spectrometryAn Agilent RapidFire 365 platform, coupled to an Agilent 6550 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) mass spectrometer, was used to perform intact protein mass spectrometry in positive ion mode with a jet-stream electrospray ion source (Agilent). Proteins with disulfide bonds were reduced with 2.5鈥塵M TCEP in 50鈥塵M Tris-HCl with 5鈥塵M EDTA at pH 8.5 for 1鈥塰 before treatment with formic acid. Protein samples at 10鈥壩糓 in 50鈥壩糒 volume were prepared on a 384-well polypropylene plate (Greiner) and acidified to 1% (v/v) formic acid. Samples were aspirated under vacuum for 0.4鈥塻 on the RapidFire sampling platform and loaded onto a C4 solid-phase extraction cartridge. Following washes with 0.1% (v/v) formic acid at 1.5鈥塵L/min flow-rate for 5.5鈥塻, the samples were eluted to the mass spectrometer with deionized water containing 85% (v/v) acetonitrile and 0.1% (v/v) formic acid at 1.25鈥塵L/min for 5.5鈥塻. The cartridge was equilibrated with deionized water for 0.5鈥塻. Nitrogen drying gas for the ionization source was operated at 13鈥塋/min at 225鈥壜癈, with the jet stream sheath gas at a flow-rate of 12鈥塋/min at 350鈥壜癈 and the nozzle voltage at 1500鈥塚. Data analysis was done using Mass Hunter Qualitative Analysis software version 7.0, with the protein ionization data deconvoluted using the maximum entropy algorithm. Predicted mass was calculated using ExPASy ProtParam, based on all disulfide bonds being reduced and cleavage of the N-terminal formylmethionine.SEC-MALSSamples were prepared at 2鈥塵g/mL in 100鈥壩糒 in 50鈥塵M Tris-HCl, 150鈥塵M NaCl pH 8.0 and injected into a Superdex 200 HR 10/30 column (GE Healthcare) at 25鈥壜癈 at a flow-rate of 0.5鈥塵L/min connected to a Shimadzu HPLC system comprising LC-20AD pump, SIL-20AC autosampler and SPD20A UV/Vis detector with 50鈥塵M Tris-HCl, 150鈥塵M NaCl pH 8.0 as running buffer. Light scattering was detected by a Wyatt Dawn HELEOS-II 8-angle light scattering detector and Wyatt Optilab rEX refractive index monitor. The resulting light scattering, refractive index and UV traces were processed in ASTRA 6 (Wyatt Technologies).Dynamic light scatteringSamples were prepared at 1鈥塵g/mL in 50鈥塵M Tris-HCl, 150鈥塵M NaCl pH 8.0 and centrifuged for 30鈥塵in at 16,900脳g at 4鈥壜癈 to remove any aggregates. 20鈥壩糒 of each sample was loaded into a reusable quartz cuvette and measurements were taken at 20鈥壜癈 using an Omnisizer (Viscotek) with 10鈥塻cans of 10 s each. Data were analyzed using OmniSIZE 3.0 and the intensity distribution from the scans were plotted in Excel.Fluorescence assayA concentration gradient of SpyTag-mClover3 in PBS, purified by either Spy Go or Ni-NTA, was prepared in triplicate from 15鈥壩糓 to 0.47鈥壩糓 in a black, flat-bottom half-area 96-well plate (Corning). The protein was excited at 位ex鈥?鈥?/sub>482鈥壜扁€?6鈥塶m and fluorescence intensity was detected at 位em鈥?鈥?30鈥壜扁€?0鈥塶m, at 40 flashes per well using a CLARIOstar plate reader (BMG Labtech) at 30鈥壜癈. The fluorescence intensity was plotted using Microsoft Excel.Cell killing assayMDA-MB-231 cells from American Type Culture Collection were grown at 37鈥壜癈鈥?with 5% CO2 in Dulbecco鈥檚 Modified Eagle Medium (DMEM) (Life Technologies) containing 10% (v/v) Fetal Bovine Serum (Sigma-Aldrich) and 50鈥塙/mL penicillin and 50鈥壩糶/mL streptomycin (Sigma-Aldrich). Cells were passaged upon 70鈥?0% confluency and for 3 months in total. MDA-MB-231 cells were seeded into a 96-well plate at 40,000 cells per well in 100鈥壜礚 DMEM containing 50鈥塙/mL penicillin and 50鈥壩糶/mL streptomycin and 1% (v/v) FBS and incubated at 37鈥壜癈 with 5% CO2 for 16鈥塰. Each SpyCatcher002-oligomer:伪DR5-SpyTag was prepared at 100鈥塶M monomeric concentration and serial dilutions thereof, in DMEM with antibiotics and 1% (v/v) FBS as above. For a negative control, SpyTag-MBP (at 100鈥塶M and serial dilutions) was added to cells. Cells were washed with sterilized PBS twice before addition of the conjugates. The cells were incubated with the protein samples at 37鈥壜癈 with 5% CO2 for 24鈥塰. Cell viability was tested by addition of 40鈥壜礚 0.15鈥塵g/mL Resazurin (Alamar Blue) (Sigma-Aldrich) in PBS at 37鈥壜癈 with 5% CO2 for 4鈥塰 and by measuring fluorescence (位ex 544鈥塶m, 位em 590鈥塶m) using a SpectraMax3 plate reader with SoftMax Pro 5.4 software (Molecular Devices). The percentage of viable cells was calculated as 100鈥壝椻€?signal of treated cells - signal without cells)/(signal untreated cells鈥搒ignal without cells). The signal without cells was taken as the resazurin fluorescence in the absence of cells, whereas the signal of untreated cells came from the fluorescence of resazurin with cells that were incubated with DMEM with antibiotics and 1% (v/v) FBS.Graphics and sequence analysisStructures are shown from SpyTag/SpyCatcher (PDB ID 4MLI)76, oDi (PDB ID: 4DZM)46, oTri (PDB ID: 4DZL)46, oTet (PDB ID: 1USE)49, oPent (PDB ID: 1VDF)50, oHex (PDB ID: 4PN9)47, oHept (2YF2)51, CC-Tet (3R4A)46, CC-Hept (4PNA)47, or PvuII (3KSK). PyMOL 2.0 was used to visualize the structure of proteins based on PDB IDs. Amino acid sequence alignment was done with Clustal Omega.SoftwareMicrosoft Excel was used to plot DLS and fluorescence graphs. GraphPad Prism v7.0 was used to plot the SEC-MALS graph. OriginPro 2015 was used to plot the cell killing assay and mass spectrometry graphs. MicroCal PEAQ-ITC Analysis Software version 1.1.0.1262 was used to plot ITC graphs.Reporting summaryFurther information on experimental design is available in the聽Nature Research Reporting Summary linked to this article. Amino acid sequences of SpyDock and SpyCatcher002-oligomers are available in the Supplementary Information. Sequences of other constructs are available in GenBank as described above under 鈥淧lasmids and cloning鈥? Plasmids encoding the SpyDock and SpyCatcher002-oligomers were deposited in the Addgene repository (https://www.addgene.org/Mark_Howarth/). The source data for Fig.聽2a, b, d, 3, 4, 5, 6b, c, 7b鈥揹, and Supplementary Figs.聽1, 3, 4, 5, 6, and 8b, c are provided as a Source Data File. Further information and request for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Mark Howarth (mark.howarth@bioch.ox.ac.uk). References1.Kimple, M. E. Sondek, J. Overview of affinity tags for protein purification. Curr. Protoc. 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Microbiol. 74, 950鈥?58 (2008).CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsFunding was provided by Yayasan Khazanah, Oxford Centre for Islamic Studies, St. John鈥檚 College Oxford (I.N.A.K.A.), the European Research Council (ERC-2013-CoG 615945-PeptidePadlock) (A.B., A.H.K., and M.H.), and the Biotechnology and Biological Sciences Research Council (BBSRC Research Experience Placements) (G.I.N.). We thank Dr. David Staunton of the University of Oxford Department of Biochemistry Biophysical Suite for assistance. We acknowledge Dr. Anthony Tumber of the University of Oxford Department of Chemistry for assistance with MS, supported by the BBSRC (grant BB/R000344/1). We thank Dr. James F. Ross (Howarth laboratory), Dr. Anne-Marie C. Andersson (Howarth laboratory), and Dr. Karthik Rajasekar (David Sherratt laboratory) for advice.Author informationAffiliationsDepartment of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UKIrsyad N. A. Khairil Anuar,聽Anusuya Banerjee,聽Anthony H. Keeble,聽Alberto Carella,聽Georgi I. Nikov聽 聽Mark HowarthAuthorsIrsyad N. A. Khairil AnuarView author publicationsYou can also search for this author in PubMed聽Google ScholarAnusuya BanerjeeView author publicationsYou can also search for this author in PubMed聽Google ScholarAnthony H. KeebleView author publicationsYou can also search for this author in PubMed聽Google ScholarAlberto CarellaView author publicationsYou can also search for this author in PubMed聽Google ScholarGeorgi I. NikovView author publicationsYou can also search for this author in PubMed聽Google ScholarMark HowarthView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsI.N.A.K.A. performed all experiments except for cellular analysis and ITC. A.B. performed cellular analysis. A.H.K. performed ITC. A.H.K., A.C., and G.I.N. performed early optimization of SpyDock reactivity. I.N.A.K.A. and M.H. designed the project and wrote the manuscript. All authors approved the manuscript.Corresponding authorCorrespondence to Mark Howarth.Ethics declarations Competing interests M.H., A.H.K., and A.C. are authors on a patent application by the University of Oxford covering sequences for enhanced isopeptide bond formation (UK Intellectual Property Office 1706430.4). M.H. is an author on a granted patent covering peptide tags forming spontaneous isopeptide bonds (EP2534484) and a SpyBiotech co-founder, shareholder, and consultant. M.H. and I.N.A.K.A. are authors on a patent application by the University of Oxford covering Spy Go (UK Intellectual Property Office 1819850.7). The remaining authors declare no competing interests. Additional informationJournal peer review information: Nature Communications thanks Sebyung Kang and the other anonymous reviewer(s) for their contribution to the peer review of this work.Publisher鈥檚 note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary information Tiong Kit Tan, Pramila Rijal, Rolle Rahikainen, Anthony H. Keeble, Lisa Schimanski, Saira Hussain, Ruth Harvey, Jack W. P. Hayes, Jane C. Edwards, Rebecca K. McLean, Veronica Martini, Miriam Pedrera, Nazia Thakur, Carina Conceicao, Isabelle Dietrich, Holly Shelton, Anna Ludi, Ginette Wilsden, Clare Browning, Adrian K. Zagrajek, Dagmara Bialy, Sushant Bhat, Phoebe Stevenson-Leggett, Philippa Hollinghurst, Matthew Tully, Katy Moffat, Chris Chiu, Ryan Waters, Ashley Gray, Mehreen Azhar, Valerie Mioulet, Joseph Newman, Amin S. Asfor, Alison Burman, Sylvia Crossley, John A. Hammond, Elma Tchilian, Bryan Charleston, Dalan Bailey, Tobias J. Tuthill, Simon P. Graham, Helen M. E. Duyvesteyn, Tomas Malinauskas, Jiandong Huo, Julia A. Tree, Karen R. Buttigieg, Raymond J. Owens, Miles W. Carroll, Rodney S. Daniels, John W. McCauley, David I. Stuart, Kuan-Ying A. Huang, Mark Howarth Alain R. Townsend Nature Communications (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.

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