to advance bottom - up 
 nanoassembly , it is important to develop building 
 blocks for modular and robust construction , able to withstand real - world 
 challenges .
the rapid and exceptionally 
 tight binding of biotin conjugates to avidin or streptavidin provides 
 a ubiquitous tool for nanoassembly . as 
 well as
( strept)avidin s ability to sensitively detect and 
 immobilize biotinylated ligands ( which have included drugs , nonbiological 
 building blocks , and all classes of biomolecule ) , ( strept)avidin clusters 
 ligands into tetramers .
this tetravalent 
 assembly gives an avidity enhancement which is valuable in diverse 
 contexts , notably for monitoring the immune response to cancer , autoimmune 
 disease , and infection .
the possibilities for nanoassembly would be 
 greatly extended by 
 a second robust interaction to ( strept)avidin .
there are a range of 
 other ligands for ( strept)avidin including biotin analogs ( e.g. , iminobiotin , 
 desthiobiotin ) , small molecule ligands dissimilar to biotin ( notably 
 4-hydroxyazobenzene-2-carboxylic acid , haba ) , 
 peptides , and nucleic acid aptamers ; however , their binding strengths 
 are all far from the femtomolar stability of biotin binding and these 
 ligands are generally released by competition with biotin .
a unique cysteine can be introduced into streptavidin for chemical 
 labeling , but it is limiting to have to work under 
 precise oxidation states for enabling reaction , while new cysteines 
 often impair folding of potential partner proteins . to address 
 this challenge we made use of spontaneous amide bond 
 formation , creating a streptavidin tetramer able to form two orthogonal 
 ultrastable interactions .
spytag is a peptide we developed which forms 
 a spontaneous isopeptide bond to its protein partner spycatcher simply 
 upon mixing .
both spytag and spycatcher 
 components are genetically encodable , can be positioned at various 
 locations in the protein chain , are reactive under a wide range of 
 conditions , and do not possess cysteines . here
we explore how spytag and spycatcher can be linked to streptavidin 
 components , how these chimeric assemblies enable assembly of complex 
 protein architectures , and how such nanoassemblies can powerfully 
 stimulate cellular signaling .
pet21-core traptavidin ( tr ) , pet21-core streptavidin ( sa ) , pet21-dead ( d ) , and pet21-dead - d - loop 
 ( dd ) have been described .
pcr was performed 
 with kod hot start polymerase ( emd millipore ) . for the addition of 
 a c - terminal hexaglutamate tag to core traptavidin to give pet21-tre 
 ( genbank kj906519 , addgene i d 59549 )
, 5-atacatatggctgaagctggtatcaccggcacctgg 
 and 5-cgcaagcttttattactcttcctcttcctcttcggaagcagcggacggtttaactttgg 
 were used for pcr , digested with ndei and hindiii , 
 and subcloned into pet21 .
pet21-dtag ( genbank kj906520 , addgene i d 
 59548 ) was generated by insertion of spytag ( lacking the terminal 
 lys ) at the c - terminus via a gly / ser 
 spacer into pet21-dead : site - directed ligase - independent mutagenesis 
 ( slim ) was performed with 5-gtgatggtggatgcctacaaacctacgtaataaaagcttgcggccgcactcg , 
 5-taataaaagcttgcggccgcactcg , 
 5-gatgtgggcgccgcctgatcctgatccggaagcagcggacggtttaactttg , 
 and 5-ggaagcagcggacggtttaactttg .
to 
 make pet21-dcatch ( genbank kj906521 , addgene i d 59547 ) , for the initial 
 insertion of spycatcher at the c - terminus with a gly / ser spacer on 
 pet21-dead d - loop , overlap extension pcr was performed .
the forward 
 primer 5-atctcatatggctgaagctggtatcacc 
 and the reverse primer 5-cctgatcctgaagcagcggacggtttaac 
 were used to amplify the dd portion , and the forward primer 5-ccgctgcttccggatcaggatcaggagattacgacatcccaacgacc 
 and the reverse primer 5-tactaagcttttaaatatgagcgtcacctttagttg 
 were used to amplify the spycatcher portion from pdest14-spycatcher .
the resulting two pcr products were mixed and 
 reamplified using the dd portion forward primer and the spycatcher 
 portion reverse primer , digested with ndei and hindiii , and subcloned 
 into pet21 . to shorten the n - terminus of the spycatcher region , to create pet21-dcatch ,
pet21-sae 
 and pet21-ap - affiigf1r with an acceptor peptide ( ap tag ) for bira - mediated biotinylation have been described 
 previously .
sa , sae , tr , tre , 
 dtag , and dcatch were expressed in e. coli bl21 [ de3 ] 
 ripl ( stratagene ) and refolded from inclusion bodies by dilution into 
 pbs in a similar method to that previously described .
after iptg induction , cells were harvested by centrifugation 
 at 4000 g. the pellet was resuspended in 10 ml of pbs and stored at 
 80 c . to isolate the inclusion bodies , 10 ml of pbs 
 ( 10.1 mm na2hpo4.2h2o , 2.7 mm kh2po4 ,
2.7 mm kcl , 137 mm nacl ph 7.4 ) containing 
 edta - free mixed protease inhibitors ( roche ) , 1 mm phenylmethylsulfonyl 
 fluoride ( pmsf ) , 1% triton x-100 , 10 mm edta , and 0.1 mg / ml hen egg 
 lysozyme ( sigma ) were added to the cell suspension .
once thawed , this 
 mixture was kept on ice for 1 h and then sonicated on ice using 4 
 30 s pulses , with 1 min of rest between each pulse .
lysed cells were 
 then centrifuged at 10  000 g for 30 min at 
 4 c , and the supernatant was discarded .
the pellet was thoroughly 
 resuspended in pbs containing 1% triton x-100 and 10 mm edta and again 
 centrifuged at 10  000 g for 30 min at 4 c . 

inclusion bodies were then washed in pbs containing 10 mm edta and 
 again centrifuged at 10  000 g for 30 min at 
 4 c .
the washed inclusion body pellet was then dissolved in 
 5 ml of 6 m guanidinium hydrochloride ph 1.0 on ice , and any insoluble 
 matter was removed by centrifugation at 10  000 g for 30 min at 4 c .
the a280 of 
 the dissolved inclusion bodies was then measured ( typically 3040 ) , 
 and an equivalent number of absorbance units of the appropriate variant 
 was mixed together ; e.g. , 5 ml of tre dissolved inclusion bodies with 
 an a280 of 30 were mixed with 3.75 ml 
 of dtag dissolved inclusion bodies with an a280 of 40 .
homotetramers sa4 , sae4 , tre4 , and dtag4 were generated 
 by refolding without mixing with any other streptavidin variant . for refolding ,
the inclusion bodies were diluted dropwise ( by gravity 
 through a 10 l pipet tip secured with parafilm to a 20 ml syringe 
 barrel ) into 200 ml of stirred precooled pbs plus 10 mm edta at 4 
 c and left stirring for 16 h. to the refolded inclusion bodies 
 we then added 60 g of ( nh4)2so4 ( 99% , 
 acros organics ) , and the mixture was stirred for 1 h at 4 c . 

this solution was then crudely filtered through several paper towels , 
 and a further 60 g ( nh4)2so4 were 
 added to the clarified solution . after a further 1 h of stirring at 
 4 c , the protein was collected by centrifugation at 15  000 g for 30 min .
the pelleted protein was then dissolved in 
 50 ml of 50 mm boric acid with 300 mm nacl ( ph adjusted to 11.0 with 
 4 m naoh ) and again centrifuged at 10  000 g for 30 min at 4 c . the clarified mixture of either tre / dtag 
 or tr / dcatch was first purified on a 5 ml iminobiotin - sepharose affinity 
 column ( affiland , s.a . ) using 50 mm sodium borate , 300 mm nacl ph 
 11.0 as the binding and wash buffer and 20 mm kh2po4 ph 2.2 as the elution buffer .
the eluate was then exchanged 
 into 20 mm trishcl ph 8.0 by 
 dialysis and loaded onto a 5 ml q - hp column ( ge healthcare ) .
the different 
 forms were isolated by using a 50 column volumes ( i.e. , 250 ml ) linear 
 gradient of 0.150.4 m nacl and collecting 5 ml fractions with 
 a 4 ml / min flow rate using 20 mm trishcl ph 8.0 as the running 
 buffer . for closely eluting peaks , sds - page sometimes showed that 
 separation was incomplete , in which case a second round of ion - exchange 
 chromatography was performed by loading samples onto a 1 ml mono - p 
 5/50 gl column ( ge healthcare ) with 1 ml / min flow rate .
separation 
 was performed by applying a 60 column volumes ( i.e. , 60 ml ) linear 
 gradient of 0.250.4 m nacl and collecting 2 ml fractions . 

the eluted fractions were concentrated to 510 mg / ml using 
 a vivaspin centrifugal concentrator 30 kda cutoff ( ge healthcare ) , 
 dialyzed into pbs , and stored at 80 c .
all purification 
 steps were performed using an kta purifier 10 ( ge healthcare ) .
glutathione - s - transferase - bira ( plasmid a kind gift from chris 
 ocallaghan , university of oxford ) was expressed in e. coli and purified using glutathione - sepharose as described .
ap - affiigf1r and affiher2-spytag were expressed 
 in e. coli and purified using ni - nta ( qiagen ) .
protein concentrations 
 were determined from a280 using the extinction 
 coefficient calculated from the amino 
 acid sequence using the protparam tool .
the pi of each monomer 
 was estimated from the sequence using the protparam tool .
the tetramer 
 pi was estimated from the weighted mean of the pi of each monomer . to generate 
 an octamer ,
40 m tr3dcatch1 and 40 m tre3dtag1 were 
 mixed together and incubated for 1 h at room temperature .
the sample 
 was then concentrated 2-fold using a centrifugal concentrator 6 kda 
 cutoff and incubated for 16 h at room temperature .
octamer was then 
 isolated by size exclusion chromatography on a superdex 200 gl 10/300 
 column ( ge healthcare ) using pbs ph 7.4 as a running buffer and a 
 flow rate of 0.5 ml / min . to generate an eicosamer , 400 m 
 tr3dcatch1 was mixed with 100 m dtag4 to give a final concentration 
 of 250 m tr3dcatch1 and 40 m dtag4 .
samples were incubated 
 for 1 h at room temperature and then concentrated 2-fold using a centrifugal 
 concentrator 6 kda cutoff and further incubated for 16 h at room temperature . 

the eicosamer was then isolated using size exclusion chromatography 
 on a superdex 200 gl 10/300 column ( ge healthcare ) using pbs ph 7.4 
 as a running buffer and a flow rate of 0.5 ml / min .
sds - page was performed on 10% , 14% , and 16% 
 tris - glycine gels using an xcell surelock system ( life technologies ) . 

protein samples ( 10 m ) were mixed with an equal volume of 2 
  sds loading buffer ( 20% glycerol , 100 mm trishcl , 4% 
 sds , 0.2% bromophenol blue , ph 6.8 ) .
variants of streptavidin and traptavidin remain 
 folded , assembled in a tetramer , and bound to biotinylated ligands 
 on sds - page without boiling ; however , since they are not linear their 
 mobility does not match the molecular weight and binding of biotinylated ligands can sometimes increase mobility . 
 also , the relative mobility of a folded tetramer depends on the polyacrylamide 
 percentage , such as for tr4 and tr3dcatch1 in figure 3c versus figure s2b .
gels were typically run for 
 1 h at room temperature at 200 v in tris - glycine running buffer ( 25 
 mm trishcl , 192 mm glycine , and 0.1% sds , ph 8.2 ) .
sds - page 
 was also performed on 4% and 7% tris - acetate polyacrylamide gels . 

nondenatured samples were loaded without boiling in 5  loading 
 buffer ( 150 mm tris - acetate ph 7.0 , 10% sds , 25% sucrose , 0.2% bromophenol 
 blue ) .
gels were typically run for 90 min on ice at 150 v in an xcell 
 surelock in running buffer ( 50 mm tricine , 50 mm trishcl , and 
 0.1% sds , ph 8.2 ) .
gels were stained with instantblue ( expedeon ) and 
 imaged using a chemidoc xrs imager and quantityone ( version 4.6 ) software 
 ( bio - rad ) .
tre3dtag1 was incubated with the indicated concentrations 
 of bio - affiigf1r 
 and spycatcher for 1 h at room temperature in pbs .
tr3dcatch1 was 
 incubated with the indicated concentration of affiher2-spytag and 
 bio - affiigf1r for 1 h at room temperature in pbs .
samples were then 
 analyzed by sds - page with coomassie staining . for testing binding 
 by mhc , we used biotinylated hla - a2 bound to ny - eso-1157165 9v ( see below ) .
proteins were incubated together overnight at room 
 temperature in pbs and then analyzed by sds - page with coomassie staining 
 ( for sa4 and tr4 a 10% polyacrylamide tris - glycine gel ; for the octamer 
 a 7% polyacrylamide tris - acetate gel ; for the eicosamer a 4% polyacrylamide 
 tris - acetate gel ) . for stability tests , tre3dtag1 or tr3dcatch1 
 at 20 m
were 
 incubated in pbs , 0.1% sodium azide , 1 mm pmsf , 1 mm edta , and edta - free 
 mixed protease inhibitors ( roche ) for 18 d at 25 or 37 c 
 before sds - page .
samples were concentrated 
 to 15 
 m and buffer - exchanged into 200 mm ammonium acetate using amicon 
 ultra 0.5 ml centrifugal filters with a 10 kda cutoff ( millipore ) . 

measurements were carried out on a modified synapt g1 high definition 
 mass spectrometry ( hdms ) quadrupole time of flight ( q - tof ) mass spectrometer 
 ( waters ) , calibrated using 10 mg / ml cesium 
 iodide in 250 mm ammonium acetate .
2.5 l aliquots of sample 
 were delivered by nanoelectrospray ionization via gold - coated capillaries , 
 prepared in house .
instrumental parameters 
 for tetramers and octamers were as follows : source pressure 6.0 mbar , 
 capillary voltage 1.20 kv , cone voltage 50 v , trap energy 10 v , bias 
 voltage 5 v , and trap pressure 0.0163 mbar .
for eicosamers , the cone 
 voltage was 100 v and the trap energy was 25 v. mass spectra were 
 smoothed and peak - centered , and masses were assigned using masslynx 
 v4.1 ( waters ) .
the 
 eicosamer , octamer , and sae4 in pbs at 10 m in a final volume 
 of 50 l were incubated for 3 min at 25 , 37 , 50 , 60 , 70 , 80 , 
 or 90 c and then cooled to 10 c in a bio - rad c1000 thermal 
 cycler at 3 c / s .
sds loading buffer was added to each sample 
 directly before loading onto sds - page . as a control ,
the eicosamer , 
 octamer , and sae4 were incubated for 3 min at 95 c in the presence 
 of sds loading buffer .
sds - page was performed for the eicosamer on 
 a 4% tris - acetate gel , octamer on a 7% tris - acetate gel , and sae4 
 on an 18% tris - glycine gel . peptide 
mhc 
 complexes were generated by expressing the hla - a2 mhc heavy chain 
 and human 2-microglobulin as inclusion bodies in e. coli and then refolding with solid - phase synthesized 
 peptide ( sigma - genosys ) .
cognate peptide 
 was ny - eso-1157165 9v , with sequence sllmwitqv 
 ( a variant of the original tumor - derived peptide sllmwitqc ) ; the control 
 peptide ( sequence glggggggv ) still had the p2 and p9 anchor residues 
 for efficient binding to hla - a2 .
the 
 heavy chain contained an ap - tag at its c - terminus and was biotinylated in vitro with bira .
the biotinylated complexes were purified 
 by fplc on a superdex-75 column ( ge healthcare ) using 50 mm trishcl 
 ph 8.0 with 150 mm nacl as the running buffer .
mhc complex was incubated with a concentration 
 of the sae4 , tre4 , octamer , or eicosamer such that there was 1.4 mhc 
 to 1 biotin binding site .
incubation was for 1 h at room temperature 
 in pbs , and then samples were stored at 4 c before t cell assay .
the human leukemia jurkat t 
 cell line j.rt3-t3.5 was stably transduced with the human 1g4 t cell 
 receptor and the human cd8 coreceptor .
the 1g4 t cell receptor has a binding affinity for the cognate peptide  mhc 
 complex of 7.2 m as measured by surface plasmon resonance .
cells were cultured in dmem with 10% fetal calf 
 serum and 1% penicillin / streptomycin and incubated at 37 c and 
 10% co2 .
t cells at 10 cells / ml in dmem 
 were incubated with an equal volume of dmem ( unstimulated samples ) 
 or the streptavidin / traptavidin constructs in dmem to give a 25 or 
 100 nm final concentration at 37 c for 3 min .
cells were then 
 fixed in 4% paraformaldehyde ( thermo fisher ) in pbs for 15 min at 
 37 c .
cells were washed twice with pbs + 1% bsa and permeabilized 
 on ice for 1 h in pbs - saponin [ pbs with 1% bsa , 0.1% saponin ( sigma - aldrich ) , 
 and 0.05% sodium azide ] . staining for phospho - erk
was performed using 
 1:100 dilution phycoerythrin - labeled anti - phosphoerk antibody [ phospho - p44/42 
 mapk ( erk1/2 ) ( thr202/tyr204 ) ( d13.14.4e ) xp rabbit monoclonal antibody , 
 catalog number # 5682 from cell signaling technology , inc . ] in pbs - saponin 
 for 1 h on ice in the dark .
cells 
 were then analyzed by flow cytometry using facscalibur and cellquest 
 pro software ( becton - dickinson ) .
10  000 cells were analyzed 
 per sample , and live cells were gated based on forward and side scatter . 

for maximal stability 
 of the core assembly , we used traptavidin rather than streptavidin . 

traptavidin is an s52 g r53d mutant of streptavidin we developed with 
 an 10-fold lower off - rate for biotin conjugates , as well as 
 greater thermostability and mechanostability , and is the most stable 
 binder of biotin conjugates .
we aimed to combine the ultrastable biotin binding of traptavidin 
 ( figure 1a ) with the covalent binding of spytag / spycatcher 
 to create spyavidins ( figure 1b ) .
( d ) subunits contain the mutations n23a , s27d , and s45a , which leads 
 to negligible biotin binding but unchanged tetramer stability . by mixed refolding from inclusion bodies of traptavidin subunits 
 and dead subunits linked to spytag ( dtag ) or spycatcher ( dcatch ) , 
 we aimed to create chimeric tetramers where each subunit could bind 
 biotin or spytag / spycatcher ( figure 1c ) .
nanohubs with 
 biotin binding and spytag / spycatcher ligation . ( a ) 
 multiple noncovalent interactions in biotin binding . the tight binding 
 of biotin ( carbons in yellow ) by traptavidin from the formation of 
 eight hydrogen bonds as well as a hydrophobic cage of tryptophans , 
 with residues mutated in traptavidin in cyan ( from pdb 2y3f ) .
( b ) spontaneous 
 isopeptide bond formation leads to a covalent linkage between a spytag - labeled 
 protein target ( cyan ) and spycatcher ( dark blue ) ( from pdb 4mls ) .
( c ) combining 
 spytag / spycatcher with traptavidin / biotin allows the formation of 
 tetramers with subunits able to bind biotin ( tr or tre ) , bind spytag 
 ( dcatch ) , or bind spycatcher ( dtag ) .
we previously showed that streptavidin tetramers containing 
 a precise 
 number of biotin binding or dead subunits could be purified by ion - exchange 
 chromatography , if there was sufficient difference in pi of the subunits .
we linked spytag to the c - terminus of d , to 
 create dtag subunits , and a tag of 6 glutamic acids to the c - terminus 
 of traptavidin , to create tre subunits ( figures 2a and s1a ) . with increasing numbers of 
 hexaglutamate tags , the tetramers required increasing amounts of nacl 
 for elution from the ion - exchange resin .
six main peaks were eluted 
 by ion exchange ( figure 2b ) . as previously , 
 we saw two distinct peaks from the different arrangement of divalent 
 tetramers ( 1,2 or 1,3 or 1,4 orientation ) ( figures 2b and s1 ) .
we verified the ion - exchange separation by sds - page , with the negatively 
 charged tags increasing tetramer mobility ( figure 2c ) .
traptavidin and the streptavidin variants in this study 
 remain as a tetramer on sds - page unless boiled .
the subunit composition 
 of the resultant tetramer is then visible by sds - page of boiled samples 
 ( figure 2c ) .
( a ) formation 
 of chimeric tetramers of traptavidin - glutamate tag ( tre ) and dead 
 streptavidin - spytag ( dtag ) by mixed refolding of inclusion bodies 
 and separation by ion - exchange chromatography . predicted pi values 
 are shown for the constituent monomers and the assembled tetramers . 

( b ) ion - exchange chromatogram showing separation of the mixed refold 
 into individual species bearing different numbers of functional biotin 
 binding sites and spytags .
a280 is plotted 
 with a solid line , and conductivity , with a dotted line .
( c ) sds - page 
 with coomassie staining of tre / dtag tetramers showing the tetramer 
 mobilities in unboiled samples ( left ) and the subunit compositions 
 in boiled samples ( right ) .
, 
 dead - d - loop subunits 
 were connected at the c - terminus through a glycine / serine linker to 
 spycatcher . given the pi of spycatcher
, we introduced the negative 
 charge onto the dead subunit by inserting multiple asp residues into 
 its 3,4 loop ( figure s1b ) .
these dcatch subunits were refolded with core traptavidin 
 ( tr ) subunits ( figure s1b and 3a ) . again
, ion - exchange 
 chromatography enabled resolution of 6 peaks ( figure 3b ) .
the tetramer forms separated by ion exchange were confirmed 
 by sds - page with or without boiling ( figure 3c ) .
tetramers containing spycatcher showed reduced gel mobility although , 
 since streptavidin variants remain globular in the unboiled gel , the 
 change in mobility was not according to molecular weight .
we found 
 that tr2dcatch2 gave two distinct gel mobilities , since in the 1,2 
 tetramer arrangement the spycatcher chains will be close together , 
 but in the 1,3 and 1,4 arrangement the two chains will be on opposite 
 sides of the tetramer ( figure s1c ) .
( a ) formation 
 of chimeric tetramer forms of traptavidin ( tr ) and dead streptavidin - spycatcher 
 ( dcatch ) by mixed refolding of inclusion bodies and separation by 
 ion - exchange chromatography . predicted pi values are shown for the 
 constituent monomers and the assembled tetramers .
( b ) ion - exchange 
 chromatogram showing separation of the mixed refold into individual 
 species bearing different numbers of functional biotin binding sites 
 and spycatchers .
a280 is plotted with 
 a solid line , and conductivity , with a dotted line .
( c ) 14% sds - page 
 with coomassie staining of tr / dcatch tetramers showing the tetramer 
 mobilities in unboiled samples ( left ) and the subunit compositions 
 in boiled samples ( right ) .
clearly , 
 many different spyavidin forms can be generated from these mixed tetramers 
 bearing dcatch or dtag .
we focused our attention on tre3dtag1 , which 
 is monovalent for spycatcher binding , and tr3dcatch1 , which is monovalent 
 for spytag binding .
affibodies are nonimmunoglobulin scaffolds , 
 selected by phage display for high affinity and selective binding . incubating tre3dtag1 with a site - specifically 
 biotinylated affibody against type i insulin - like growth factor receptor 
 ( igf1r ) , we observed three major shifts 
 in mobility when adding substoichiometric affibody concentrations 
 ( lane 5 , figure 4a ) and complete occupancy 
 of the three biotin - binding sites with a small excess of affibody 
 ( lane 6 , figure 4a ) . an extra shift upward 
 was seen when spycatcher was also added ( lane 7 , figure 4a ) , validating the bifunctional reactivity of tre3dtag1 .
( a ) 
 sds - page with coomassie staining of tre3dtag1 , showing the reactivity 
 of dtag with free spycatcher and the ability of the tre subunits to 
 bind up to three biotinylated affibodies ( bio - affiigf1r ) .
( b ) sds - page 
 with coomassie staining of tr3dcatch1 , showing the reactivity of dcatch 
 with a spytag - bearing affibody ( affiher2-spytag ) and the ability of 
 the tr subunits to bind up to three biotinylated affibodies ( bio - affiigf1r ) .
similarly , we saw three major 
 bands when adding substoichiometric 
 levels of biotinylated anti - igf1r affibodies to tr3dcatch1 ( figure 4b ) .
we further found a single shift upward , corresponding 
 to binding of one spytag - linked affibody of a separate specificity , 
 against the cancer - associated tyrosine kinase her2 ( figure 4b ) , so validating the reactivity 
 of tr3dcatch1 .
minor bands are also seen in figure 4a and 4b that are likely to correspond 
 to the altered mobility depending on whether the biotinylated ligand 
 binds a traptavidin subunit proximal or distal ( figure s1c ) to the spytag- or spycatcher - linked subunit .
we also confirmed that the spyavidin tetramers did not rearrange 
 subunit composition over time , consistent with previous traptavidin 
 tetramers : no new band appeared after 
 8 days of incubation at 37 c for tre3dtag1 ( figure s2a ) or tr3dcatch1 ( figure s2b ) .
( strept)avidin 
 is an obligate tetramer , and the vast majority of its applications 
 have had a maximal valency of four , but higher order valency may enable 
 new possibilities .
our generation of tr3dcatch1 and tre3dtag1 provided 
 a facile route to a streptavidin octamer ( 8 subunits ) , by mixing these 
 two forms ( figure 5a ) .
octamer formation was 
 validated by sds - page ( figure 5b ) . to illustrate 
 the progress of reaction
, we show incubation at an early time point 
 ( lane 3 , figure 5b ) , but the final preparation , 
 obtained after overnight reaction , was efficiently purified from any 
 remaining tetramer by gel filtration chromatography ( lane 4 , figure 5b ) .
native mass spectrometry , using a quadrupole 
 time - of - flight mass spectrometer , enabled 
 us to analyze the octameric assembly : octamer expected mass = 121  116 
 da ( with one isopeptide bond ) ; octamer observed mass = 121  223 
  16 da ( 107 da or 0.088% discrepancy to the expected mass ) ( figure 5c ) .
( b ) reaction of tre3dtag1 
 with tr3dcatch1 to generate an octamer ( lane 3 ) , which can subsequently 
 be purified via gel filtration ( lane 4 ) , analyzed by sds - page with 
 coomassie staining .
peaks corresponding 
 to the octamer are marked with circles , along with the charge state 
 of the highest peak .
( d ) reaction of dtag4 and tr3dcatch1 to generate 
 an eicosamer , which can subsequently be purified via gel filtration 
 ( far right lane ) , analyzed by sds - page with coomassie staining .
peaks corresponding to an eicosamer 
 and a hexadecamer impurity are marked with circles , along with the 
 charge state of the highest peak for each .
building further , with dtag4 at the heart , mixing with tr3dcatch1 
 should lead to an eicosamer ( 20 streptavidin subunits ) ( figure 5a ) .
tris - acetate gels enable analysis in this high molecular weight range , 
 although mobility deviates from molecular weight because streptavidin - derived 
 subunits remain folded ( figure 5d ) .
again proximal 
 or distal orientations of ( dtag4)1(tr3dcatch1)2 are likely to generate 
 distinct bands on sds - page ( figure 5d ) .
mass 
 spectrometry measurement was consistent with formation of the desired 
 eicosamer : eicosamer expected mass = 316  767 da ( with four 
 isopeptide bonds ) ; eicosamer observed mass = 318  523  
 51 da ( 1756 da or 0.55% discrepancy to the expected mass ) ( figure 5e ) . from analysis of these large noncovalent assemblies 
 by mass spectrometry
, where complete removal of water and buffer ions 
 is challenging , these values represent a good match between observed 
 and expected . both sds - page and mass spectrometry 
 detected an impurity of some hexadecamer ( 16 subunits ) ( figure 5d , e ) .
the robustness of the eicosamer and 
 octamer assemblies was tested 
 by a thermostability assay , which revealed stability comparable to 
 the wild - type streptavidin tetramer .
the 
 eicosamer and octamer remained intact up to 70 c but started 
 to rearrange at 80 c ( figure s3 ) .
mhc class i on the cell surface enables cytotoxic t cells to monitor 
 the events taking place inside the cell , to detect viral infection 
 or tumorigenesis .
mhc class i is a trimeric complex of a heavy chain 
 bound to 2-microglobulin and loaded with an 810 
 amino acid peptide ( figure 6a ) .
binding of 
 peptide  mhc complexes to the t cell receptor , for t cell activation , 
 typically has a half - life of seconds .
therefore , tetramerization of 
 peptide  mhc by streptavidin conferred stable binding to t cells 
 and led to a revolution in the ability to monitor specific immune 
 responses in infectious disease and cancer .
we initially validated binding of peptide  mhc complexes to 
 different biotin - binding assemblies , using sds - page .
the steric clash 
 from binding of the peptide  mhc complex to adjacent biotin 
 binding sites makes it hard to achieve complete occupancy of 4 mhc 
 around one streptavidin ( figure s4a ) .
however , 
 the extra stability of traptavidin s biotin binding led to 
 more uniform and high occupancy complexes with mhc ( figure s4b ) .
we also analyzed binding of mhc to the octamer 
 and eicosamer by sds - page , although with so many intermediate stoichiometries 
 and spatial orientations it is not possible to confirm the identity 
 of each band ( figure s4c and s4d ) .
to see how higher order clustering might change signal transduction , 
 we determined the activation of t cell signaling caused by a given 
 concentration of the biotinylated peptide  mhc complex bound 
 to the streptavidin , traptavidin , octamer , or eicosamer .
figure 6a illustrates the potential clustering from binding 
 of mhc class i to the eicosamer .
t cell activation was measured from 
 the phosphorylation of extracellular signal - related kinase ( erk ) , 
 an early downstream event following t cell receptor activation , as 
 assessed by intracellular flow cytometry . as a negative control ,
we used a glycine - rich peptide with efficient 
 binding to mhc but minimal binding to the t cell receptor . with 25 
 nm cognate peptide 
mhc , the streptavidin tetramer and traptavidin 
 tetramer gave a similar slight degree of activation ( figure 6b ) .
however , activation was stronger with the octamer , 
 while the eicosamer managed to activate a major fraction of the t 
 cells ( figure 6b ) . with 100 nm cognate peptide  mhc , 
 the streptavidin tetramer and traptavidin tetramer were able to activate 
 a higher proportion of t cells than at 25 nm , but the activation with 
 the octamer was superior to tetramers and was greater still with the 
 eicosamer ( figure 6b ) . importantly , none of 
 the assemblies activated the cells when bound to the control peptide , 
 even at 100 nm ( figure 6c ) . therefore , activation 
 depended on t cell receptor recognition and not any other feature 
 of the assembly , such as binding to the coreceptor cd8 in a t cell 
 receptor - independent fashion .
( a ) 
 cartoon of how an eicosamer may bind multiple biotinylated peptide  mhc 
 complexes .
( b ) t cell activation determined by flow cytometry of erk 
 phosphorylation , unstimulated or after stimulation with 25 nm ( left 
 panel ) or 100 nm ( right panel ) cognate peptide 
mhc incubated 
 with the streptavidin tetramer ( sae4 ) , traptavidin tetramer ( tre4 ) , 
 octamer , or eicosamer .
the percentage of cells with staining 
 above the threshold ( dotted line ) is marked .
( c ) t cell stimulation 
 as in ( b ) but with control peptide  mhc .
here we have generated spyavidin hubs , tetramers 
 with a defined 
 number of orthogonal binding sites for biotin or spytag / spycatcher . 

these hubs can then be linked to create large modular protein architectures , 
 including the octamers or eicosamers which were able to achieve highly 
 sensitive and specific t cell activation .
the extra stability 
 of biotin  conjugate binding by traptavidin 
 compared to streptavidin is highlighted 
 by the difficulty in assembling four biotinylated peptide  mhc 
 complexes around streptavidin .
streptavidin binding is exceptionally 
 strong for biotin itself and for small biotin conjugates , but when 
 linking large ligands , especially at adjacent binding sites in the 
 tetramer , binding efficiency declines and the extra stability of traptavidin is shown here to be valuable . 

similarly spytag / spycatcher interaction is slower and requires more 
 ligand excess when bringing together large ligands around a single 
 hub ( e.g. , in generating eicosamers ) .
this reflects a general challenge 
 in nanoassembly and points to the importance of starting with reactions 
 that are highly efficient at a small size range to be able to tolerate 
 the challenges of building on the larger scale .
sophisticated 
 assemblies can be made with dna nanotechnology , although 
 stability is often limited if integrity depends solely on short regions 
 base pairing and synthesis is generally 
 on the microgram scale .
oligonucleotides 
 are frequently biotinylated to enable ( strept)avidin linkage , but 
 it has not been straightforward to bridge precisely from this ( strep)avidin 
 to a subsequent protein .
coiled - coils or modular repeat proteins enable 
 impressive polypeptide nanostructures , but without disulfides 
 the binding affinity is often moderate and these structural elements 
 do not share the extensive infrastructure of biotinylated ligands 
 accessible to spyavidin . spyligase peptide 
peptide ligation 
 is an alternative route to connect irreversibly proteins into polymers , 
 via isopeptide bonds , but it is not yet possible to control polymer 
 size .
we anticipate that spyavidins hubs 
 will be valuable in interfacing with a range of different nanoassembly 
 scaffolds .
we have demonstrated spontaneous isopeptide bond formation 
 to two other peptide tags , and so in 
 future work it should be possible to construct more orthogonal isopeptide - based 
 linkages from streptavidin subunits .
streptavidin tetramers 
 have previously been linked together through 
 chemical coupling but without control over subunit identity or full 
 characterization of the multimers obtained . peptide 
mhc complexes can also be pentamerized with coiled 
 coils or covalently coupled to dextran ( although with heterogeneous 
 valency ) .
such multimers have shown enhanced 
 sensitivity compared to tetramers for staining of low affinity t cell 
 receptors , notably those against mhc class i bound to self - peptides 
 in autoimmune disease and antitumor responses .
therefore , eicosamers may have value in identification , isolation 
 for immunotherapy , or depletion of such t cells .
the 
 use of desthiobiotinylated mhc should also allow this multimerization 
 to be reversible , using free biotin as a competitor .
multivalent recognition using eicosamers may also help 
 to identify binding partners for other fast off - rate interactions , 
 including protein 
protein interactions in leukocyte adhesion or malarial erythrocyte invasion , and protein  carbohydrate interactions 
 in cancer metastasis .
it is still 
 an active area of investigation how engagement of the 
 t cell receptor leads to initiation of the tyrosine kinase cascade 
 and activation of the t cell . in the absence of a 
 cell  cell contact
, it is known that peptide  mhc tetramers 
 can activate t cell signaling , whereas single peptide 
. therefore , local regions 
 of t cell receptor clustering , giving local exclusion of transmembrane 
 phosphatases , may be sufficient for signal activation .
the extra clustering 
 with eicosamers may be able to prolong surface interactions with the 
 t cell receptor and also generate a larger area of phosphatase exclusion , 
 leading to more efficient downstream signal transduction . in future 
 work
it will be valuable to explore this nanoscale control of signal 
 activation , to enhance the cellular response 
 to a range of biotinylated macromolecular and small - molecule stimuli .
 the capture of biotin 
 by streptavidin is an inspiration for supramolecular 
 chemistry and a central tool for biological chemistry and nanotechnology , 
 because of the rapid and exceptionally stable interaction . 
 however , 
 there is no robust orthogonal interaction to this hub , limiting the 
 size and complexity of molecular assemblies that can be created . here 
 
 we combined traptavidin ( a streptavidin variant maximizing biotin 
 binding strength ) with an orthogonal irreversible interaction . 
 spytag 
 is a peptide engineered to form a spontaneous isopeptide bond to its 
 protein partner spycatcher . 
 spytag or spycatcher was successfully 
 fused to the c - terminus of dead streptavidin subunits . 
 we were able 
 to generate chimeric tetramers with n 
 ( 0  
 n  4 ) biotin binding sites and 4-n spytag or spycatcher 
 binding sites . 
 chimeric spyavidin tetramers bound precise numbers 
 of ligands fused to biotin or spytag / spycatcher . mixing 
 chimeric tetramers 
 enabled assembly of spyavidin octamers ( 8 subunits ) or eicosamers 
 ( 20 subunits ) . 
 we validated assemblies using electrophoresis and native 
 mass spectrometry . 
 eicosameric spyavidin was used to cluster trimeric 
 major histocompatibility complex ( mhc ) class i:2-microglobulin : peptide complexes , generating an assembly with up 
 to 56 components . 
 mhc eicosamers surpassed the conventional mhc tetramers 
 in acting as a powerful stimulus to t cell signaling . 
 combining ultrastable 
 noncovalent with irreversible covalent interaction , spyavidins enable 
 a simple route to create robust nanoarchitectures . 
Introduction
Experimental Section
Results
Discussion