Supporting Information

A network of Spies: synthesis of bioactive protein hydrogels by genetically-encoded SpyTag-SpyCatcher chemistry

Fei Sun*†, Wen-Bin Zhang*†, Alborz Mahdavi*, Frances H. Arnold*‡ and David A.

Tirrell*‡

*Division of Chemistry and Chemical Engineering, California Institute of Technology,

Pasadena, California 91125, United States

†F.S. and W.-B.Z. contributed equally to this work.

‡To whom correspondence should be addressed:

Division of Chemistry and Chemical Engineering, California Institute of Technology,

Pasadena, California 91125, United States

Phone: 626-3954162; Email: frances@cheme.caltech.edu

Division of Chemistry and Chemical Engineering, California Institute of Technology,

Pasadena, California 91125, United States

Phone: 626-3953140; Email: tirrell@caltech.edu

Sup

Fig.situ rad/

pporting Fig

. S1. Evolutgelation at

sec and 5%

gures

tion of storat 25°C. Sh, respectivel

age (G’) anear frequenly.

nd loss (G”)ncy and stra

) moduli as ain amplitu

a function ude were he

of time dureld constan

ring in nt at 1

Fig.MeaMeafreqand 12.6high

. S2. Frequasurements asurements quency decre

loss moduli6 to 0.01 rah noise level

uency sweeon AAA+

on AA’A+Beased from i, G’ and G

ad/sec; measls.

ep tests on+BB (10 wBB (10 wt%100 to 0.0”, were recosurements o

AAA+BB wt%) were

%) and AA+1 rad/sec anorded. Dataof G” at hig

(a), AA’Ae performe

+BB (10 wt%nd strain waa are shown gher freque

A+BB (b) aed at both%) were donas fixed at only for the

encies were

and AA+Bh 25 and ne at 25°C. 5%. Both se frequencycharacteriz

B (c). 37°C. Shear

storage y range zed by

Fig.MeaMeastrailoss were

. S3. Straiasurements asurements in increasedmoduli, G

e characteri

in sweep ton AAA+

on AA’A+Bd from 0.1%’ and G”, wzed by high

tests on A+BB (10 wBB (10 wt%

% to 100%; fwere recordeh noise level

AAA+BB (wt%) were

%) and AA+frequency wed. Data acls.

a), AA’A+e performe

+BB (10 wt%was fixed at quired at st

+BB (b) aed at both%) were don10 rad/sec.

train amplit

and AA+BBh 25 and ne at 25°C. Both storag

tudes below

B (c). 37°C. Shear

ge and w 0.8%

Fig.

Fig.mola(2M

. S4. Large-

. S5. Schemar ratio o

MAAA+3MBB)

-amplitude o

matic of a 10of 2:3. T)/2.

oscillatory s

00% crossliThe molec

shear tests o

inked Spy ncular weig

of the Spy ne

network comht betwee

etwork at 10

 

mprised of An crosslin

0 rad/sec.

AAA and Bnks, Mc,

BB at a equals

Fig.

. S6. Swellinng of the Sp

py network ((AAA+BB)) in water (toop) and PBSS buffer (boottom).

Fig.mChstraiat 25fromcharnetwincrechar

. S7. Rheolherry-Spy-nin amplitude5°C and 37

m 100 to 0.racterized bywork at 25°Ceased from racterized by

logy of mChnetwork at 2e at 5%. (b)°C. The she.01 rad/sec.y high noisC and 37°C0.1 to 100%

y high noise

herry-Spy-n25°C. The s) Frequency ear strain am. Measureme levels. (c)

C. The shear%. Measuree levels.

network. (a)shear frequesweep tests

mplitude waments of G”) Strain swer frequency

ements of G

) Time sweency was hes on 6 hour-as set at 5%” at frequeneep tests ony was set at G” at strain a

ep tests of ield constant-cured mCh

%. The shearncies aboven 6 hour-cur

10 rad/secamplitudes b

in situ gelatt at 1 rad/seerry-Spy-ne

r frequency e 20 rad/secred mCherry. The shear below 0.5%

tion of ec; the etwork varied

c were y-Spy-

strain % were

Fig.encaSpy-Chaare o

. S8. Confoapsulated in-network (r

annel 2 (Ch2overlays of

ocal fluorescn the mCherred), in whi2) images sthe matrix a

cence z-slicrry-Spy-netwich dark arshow two spand cells (Sc

ce micrograpwork. Channreas corresppreading 3Tcale bars, 10

phs of mounel 1 (Ch1)

pond to regiT3/GFP cel0 µm).

use NIH 3T3) images revions occupils (green).

3/GFP fibroveal the mCied by 3T3Ch1+Ch2 i

oblasts Cherry-

cells. mages

Fig.Spy-watethe tat 1 netwfrom25°C0.1 thigh

. S9. Rheolo-network ater. BB (10 wtwo precursrad/sec; str

work at 25°Cm 100 to 0.C. The sheato 100%. Mh noise level

ogy of LIF-t 25°C. AAAwt%) was d

sors in a morain amplituC. The shea025 rad/sec

ar frequencyMeasurement

ls.

-Spy-networA (10 wt%)dissolved inolar ratio of ude at 5%. (bar strain amc. (c) Strainy was set at ts of G” at s

rk. (a) Time) was dissol

n mESC me2:3 (AAA:Bb) Frequenc

mplitude wasn sweep tes

10 rad/sec.strain amplit

e sweep teslved in an A

edium. GelaBB). Shearcy sweep tes set at 5%.sts of 6 hou The shear tudes below

sts of in situA-LIF-A soation was in

frequency wsts of 6 hou. The shear ur-cured LIFstrain ampl

w 0.8% were

u gelation oolution in dinitiated by mwas held cour-cured LIF

frequency F-Spy-netwlitude variede characteriz

of LIF-istilled mixing onstant F-Spy-varied

work at d from zed by

Fig.netw

. S10. Illustrwork gel.

ration of brright-field immages acqui

ired from ddifferent regions of eachh Spy-

Fig.

. S11. Full-sscan images from immuunoblotting experimentts.

a. AAA

MKGSSHHHHH HVDAHIVMVD AYKPTKLDGH GVGVPGVGVP GVGVPGEGVP GVGVPGVGVP GVGVPGVGVP GEGVPGVGVP GVGVPGVGVP GVGVPGEGVP GVGVPGVGEL AHIVMVDAYK PTKTSVPGVG VPGVGVPGEG VPGVGVPGVG VPGVGVPGVG VPGEGVPGVG VPGVGVPGVG VPGVGVPGEG VPGVGVPGVG VPGGLLDAHI VMVDAYKPTK LEWKK

b. AA’A

MKGSSHHHHH HVDAHIVMVD AYKPTKLDGH GVGVPGVGVP GVGVPGEGVP GVGVPGVGVP GVGVPGVGVP GEGVPGVGVP GVGVPGVGVP GVGVPGEGVP GVGVPGVGEL AHIVMVAAYK PTKTSVPGVG VPGVGVPGEG VPGVGVPGVG VPGVGVPGVG VPGEGVPGVG VPGVGVPGVG VPGVGVPGEG VPGVGVPGVG VPGGLLDAHI VMVDAYKPTK LEWKK Fig. S12. Amino acid sequences of (a) AAA and (b) AA’A. Reactive Asp residues are shown in the boxes. The segments in red are SpyTags. In AA’A, the reactive Asp residue is mutated to Ala (grey). The sequences of AA and BB have been published (1).

A-mCherry-A

MKGSSHHHHH HVDAHIVMVD AYKPTKLDGH GVGVPGVGVP GVGVPGEGVP GVGVPGVGVP

GVGVPGVGVP GEGVPGVGVP GVGVPGVGVP GVGVPGEGVP GVGVPGVGEL VSKGEEDNMA

IIKEFMRFKV HMEGSVNGHE FEIEGEGEGR PYEGTQTAKL KVTKGGPLPF AWDILSPQFM

YGSKAYVKHP ADIPDYLKLS FPEGFKWERV MNFEDGGVVT VTQDSSLQDG EFIYKVKLRG

TNFPSDGPVM QKKTMGWEAS SERMYPEDGA LKGEIKQRLK LKDGGHYDAE VKTTYKAKKP

VQLPGAYNVN IKLDITSHNE DYTIVEQYER AEGRHSTGGM DELYKTSVPG VGVPGVGVPG

EGVPGVGVPG VGVPGVGVPG VGVPGEGVPG VGVPGVGVPG VGVPGVGVPG EGVPGVGVPG

VGVPGGLLDA HIVMVDAYKP TKLEWKK

Fig. S13. Amino acid sequence of A-mCherry-A. Reactive Asp residues are shown in the boxes. The segments in red and in yellow are SpyTags and mCherry, respectively.

A-L

MKGSGVGVTCAIHANGCRLCVGVPVGVP Fig.the b

Sup

Calc

a

b

whemod

The

c

whe×10solvpolywatenetwthe sagre

LIF-A

SSHHHHH HVVPGVGVP GEIRHPCHG NLGTEKAKL VECNKYRVG HVPGEGVPG VGPGVGVPG GL

. S14. Aminboxes. The

pporting Dis

culation of c

a. Moleculcrosslink

Mc = (3M

b. Molecul

Mc’ = ρR

ere ρ is the pdulus (0.3 kP

estimated c

c. Molecul From Fl

ere V2m = q-1

0-3 g/cm3); Vvent interacty(N,N-dimeter at 25 °C, work, we useswelling rateement with

VDAHIVMVD EGVPGVGVP LMNQIKNQL ELYRMVAYL VDVPPVPDH GVPGVGVPG LLDAHIVMV

no acid sequsegments co

scussion

crosslink de

lar weight bking):

MBB+2MAA

lar weight b

RT/G = 826

polymer denPa).

crosslinking

lar weight b

lory network

1 (q = swelliV1 is the moion parametthylacrylamχ1 = ~ 0.5 (3ed a value otio, q, is 310h the value o

AYKPTKLDGGVGVPGVGVAQLNGSANASASLTNITRSDKEVFRKKVGVPGVGVPDAYKPTKLE

uence of A-Lolored in ye

ensities from

between cros

AA)/2 = 91.5

between cros

6 kDa

nsity (0.100

g efficiency

between cros

k theory (2)

ing ratio); ρolar volumeter. For hyd

mide), poly(N3). Althougof 0.5 to esti0, and a valuof Mc calcul

GH GVGVPGVVP GVGVPGEAL FISYYTARD QKVLNPSKK LGCQLLGPG EGVPGVGEW KK

LIF-A. Reacellow and in

m storage mo

sslinks of th

kDa

sslinks dedu

g/cm3 for a

(Mc/Mc’) =

sslinks dedu

):

ρ2 is the poly of solvent (

drophilic polN-isopropylh χ1 is not kimate Mc froue of 0.5 forated from th

VGVP GVGVPEGVP GVGVPAQGE PFPNNSAVS LHSKLGTYK QVISVGVPG VGVPG

ctive aspart red are LIF

odulus G’ an

he perfect Sp

uced from th

a 10 wt% ne

11%.

uced from th

ymer density(18 cm3/molymers suchlacrylamideknown for thom the swelr χ1 gives Mhe storage m

PGEGVP GVGPGVGEL SPLNLDKLC GPNLNATID VMRVVVQAF TSVGVGVPG VGV

tic acid residF and SpyTa

nd swelling

py network

he storage m

twork) and

he swelling

y after swel

ol); and χ1 ish as polyacry), and poly(

he proteins tlling data. Inc = ~750 kD

modulus.

GVPGVGVP LPITPVNA NVTDFPPF RGLLSNVL VPGVGVPG VPGEGVPG

dues are shoag, respectiv

g ratio q:

(100%

modulus:

G is the sto

ratio in wat

lling in wates the polymeylamide, (vinyl alcohthat form thn distilled w

Da, in reason

60 120 180 240 300 360 392

own in vely.

orage

ter:

er (3.2er-

hol) in e Spy

water, nable

The crosslink densities derived from the storage modulus and the swelling ratio are approximately ten-fold lower than that of the perfect Spy network, indicating incomplete crosslinking.

Table S1. Bacterial strains，plasmids, and primers used in this study.

Strains Relevant Characteristics Source

E. coli

DH5α

Stratagene

BL21 star(DE3)

Invitrogen

Plasmids Relevant Characteristics Source

pQE-80L T5 promoter-operator, N-terminal His tag, Ampr

Qiagen

pQE-ELP pQE-80L plasmid containing the gene encoding elastin

Starting vector for all recombinant proteins in this study

pQE-EA Plasmid for expression of Elastin-SpyTag

This study

pQE-EB Plasmid for expression of Elastin-SpyCatcher

This study

pQE-AA Plasmid for expression of SpyTag-Elastin-RGD-Elastin-SpyTag

This study

pQE-BB Plasmid for expression of SpyCatcher-Elastin-RGD-Elastin-SpyCatcher

This study

pQE-AAA Plasmid for expression of SpyTag-Elastin-SpyTag-Elastin-SpyTag

This study

pQE-AA’A pQE-AAA-D117A mutant, where the internal SpyTag is inactivated by Asp117 to Ala mutation

This study

pQE-A-mCherry-A Plasmid for expression of SpyTag-Elastin-mCherry-Elastin-SpyTag

Primers Sequence

SpyCatcher-SalI-F GTGTACA GTCGAC A TC CCA ACG AC C GAA AAC CTG TATTTTC

spyCatcher-XhoI-R GTAGGCACTCGAG CTGACCCCAAATACCTTGCGGACCGTC

SpyTag-SalI-XhoI-F GACA GTCGACGCCCATATTGTCATGGTTGATGCATACAAGCCGACGAAGCTCGAG GTAGG

SpyTag-SalI-XhoI-R CCTACCTCGAGCTTCGTCGGCTTGTATGCATCAACCATGACAATATGGGCGTCGACTGTC

SpyTag-SacI-SpeI-F GACA GAGCTCGCCCATATTGTCATGGTTGATGCATACAAGCCGACGAAGACTAGT GTAGG

SpyTag-SacI-SpeI-R CCTACACTAGTCTTCGTCGGCTTGTATGCATCAACCATGACAATATGGGCGAGCTCTGTC

SpyTagD20A-SacI-SpeI-F

GACA GAGCTC GCCCATATTGTCATGGTTGCGGCATACAAGCCGACGAAGACTAGT GTAGG

SpyTagD20A-SacI-SpeI-R

CCTACACTAGTCTTCGTCGGCTTGTATGCCGCAACCATGACAATATGGGCGAGCTCTGTC

mCherry-SacI-F ATAA GAGCTC ATGGTGAGCAAGGGCGAGG

mCherry-SpeI-R CAA TAC TAG TCT TGT ACA GCT CGT CCA TGC CGT C

LIF-SacI-F GTAC GTCGAC GAGCTC TCT CCG CTG CCG ATC ACC CCG GTA AAC GCA ACC TGT GCC ATC C

LIF-SpeI-R GTAGCTCGAGACTAGTAAACGCCTGAACAACCACAGAGATAACCTGTTTGTAAGTAC

 

 

References

1. Zhang WB, Sun F, Tirrell DA, & Arnold FH (2013) Controlling macromolecular topology with genetically encoded SpyTag-SpyCatcher chemistry. J Am Chem Soc 135(37):13988-13997.

2. Sperling LH (2006) Introduction to Physical Polymer Science, 4th edition, pp. 472-473. Wiley & Sons. ISBN: 978-0-471-70606-9.

3. Orwell RA and Arnold PA (2006) Chapter 14. Polymer-Solvent Interaction Parameter χ.In Mark JE (ed.) Physical Properties of Polymers Handbook, 2nd edition, pp. 233-256. Springer. ISBN: 978-0-387-31235-4.