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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.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-
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.