Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451
Weinheim, 2009
DOI: 10.1002/cbic.200900542
Supporting Information
for
Generation of Surface-Bound Multicomponent Protein
Gradients
Kechun Zhang, Ayae Sugawara, and David A. Tirrell*
Cloning, expression and purification of proteins.
Genes encoding the elastin mi-
metic domain ELF and leucine-zipper peptides ZE and
ZR were constructed previ-
ously.
[1]
Because ZRELF is more soluble than the ZE(gs)
6
ELF scaffold we reported in
ref [1], we prefer ZRELF for protein immobilization
. ZRELF containing
p
-azidophenyl-
alanine was expressed in phenylalanine auxotrophic
E. coli
strain AF-IQ.
[2]
Gene
fragments encoding FNZE and QKZE were ligated into
pQE60 and the resulting plas-
mids were transformed into
E. coli
strain BL-21. Protein expression in 2XYT medium
was induced by 1 mM IPTG at 22
o
C. FNZE and QKZE were purified on Ni-NTA col-
umns; purity of all expressed proteins was assessed
by SDS-PAGE and MALDI-TOF
mass spectrometry. Protein sequences are shown belo
w:
ZRELF
MKGS
LEIRAAALRRRNTALRTRVAELRQRVQRLRNEVSQYETRYGPL
GGGGSGGGGSG
[(VPG
VG)
2
VPG
F
G(VPGVG)
2
]
5
VPGC
FNZE
MKGSKK
VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVP
GSKSTATISGL
KPGVDYTITVYAVTPRGDWNEGSKPISINYRT
TSGS
LEIEAAALEQENTALETEVAELEQEVQRLENIVS
QYRTRYGPL
GGGRSHHHHHH
QKZE
MKGS
KLTWQELYQLKYKGI
GGGGSGS
LEIEAAALEQENTALETEVAELEQEVQRLENIVSQYRTRYGP
L
GGGRSHHHHHH
The ZR sequence is shown in green, ELF in red, Fn i
n orange, ZE in blue, and
QK in purple.
Surface functionalization.
Standard glass slides (Corning) were immersed in co
n-
centrated H
2
SO
4
for 1 h. After washing thoroughly with water, the
y were immersed in
a boiling solution of H
2
O
2
(30%) / NH
4
OH (30%) / H
2
O (1:1:5
v/v
) for 30 min and gent-
ly shaken in 1% octyltrichlorosilane in toluene for
3 h. Finally they were rinsed in
MeOH (2x) and in deionized water (2x). Functionaliz
ed slides were cured at 110 °C
for 30 min.
A solution of ZRELF (50 μL, 2.5 mg/mL) in 50% propa
n-1-ol was applied to a
glass slide and spun at 1,500 rpm for 45 s. The res
ulting protein film was irradiated
by Hg-arc lamps (I-line and H-line, 4.5 mW cm
-2
) in a Karl Suss mask aligner for 2
min. Protein-coated slides were washed thoroughly w
ith 50% isopropanol and doubly
distilled H
2
O to remove uncrosslinked protein.
Fabrication of microfluidic chips.
The microfluidic gradient generator was fabricat-
ed by rapid prototyping and soft lithography as ori
ginally described by Jeon et al.
[3]
Briefly, a high-resolution printer was used to gene
rate a mask with a minimum feature
size of 30 μm from a CAD file (CAD/Art Services, Po
way, CA). A SU-8 2100 photo-
resist (Microchem, Newton, MA) layer was spin-coate
d onto a silicon wafer at 3500
rpm for 30 s and exposed to ultraviolet light for 1
50 s through the mask in a Karl Suss
mask aligner. The wafer was then immersed into SU-8
developer, and the unexposed
photoresist was dissolved, leaving behind a master
mold composed of 100-μm high
crosslinked photoresist structures. Poly(dimethylsi
loxane) (PDMS) chips were formed
by curing prepolymer solution (Sylgard 184, Dow Cor
ning) on silicon masters. Inlet
and outlet ports were punched out of the PDMS using
a sharpened needle. Polyeth-
ylene tubing was inserted into the ports to enable
fluid flow into and out of the micro-
channels.
Measurement of protein surface density
. Lyophilized samples of FNZE or QKZE
(1 mg each) were dissolved in 0.5 mL of NaHCO
3
buffer (100 m
M
, pH~9, adding SDS
until dissolution) and treated with 0.5 mg of the N
HS esters of either Cy3 (Amersham,
for FNZE) or Alexa 647 (Invitrogen, for QKZE) for 5
h at room temperature. Dialysis
was used to remove unconjugated dye. Protein soluti
ons (2 μL each) at different con-
centrations (0.2, 0.1 and 0.05 μ
M
of FNZE; 0.4, 0.2 and 0.1 μ
M
of QKZE) were spot-
ted onto ZRELF-coated glass slides and dried in air
overnight. The fluorescence in-
tensity and area of each spot were measured by a Ge
nePix 4200A chip reader. The
surface density at each spot was calculated and plo
tted against fluorescence inten-
sity.
Generation of immobilized protein gradients.
The PDMS chip and glass substrate
(with the ZRELF-coated region covered
[4]
) were activated with oxygen plasma (200
millitorr, 35 s, 80 W, Anatech) and coupled immedia
tely to form an irreversible seal.
To remove trapped bubbles and block non-specific pr
otein adsorption, microfluidic
channels were flushed with 2% BSA solution for 30 m
in using a PHD 2000 syringe
pump (Harvard Apparatus). Protein solutions in 2% B
SA were injected into the mixer
inlets at a rate of 0.5 μL/min for 1 h to generate
immobilized protein gradients. The
assembly was then soaked in water and the PDMS dire
ctly above the exit channel
was cut away with a razor blade, leaving the protei
n gradient positioned within a
PDMS well on the glass slide. The sample was sonica
ted in PBS for 30 min and
blown dry, and the gradient profile was scanned by
a GenePix 4200A chip reader.
For substrates used for cell studies, PBS was left
in the PDMS well to prevent dehy-
dration of the gradient surface.
Cell culture.
Human umbilical vein endothelial cells (HUVECs, Clo
netics) were main-
tained in a 37°C, 5% CO
2
, humidified environmental chamber. The cells were
grown
in endothelial cell basal medium (EBM-2, Clonetics)
supplemented with the supplied
Bulletkit, which was replaced every 2 days. Near-co
nfluent HUVEC cultures were
passaged nonenzymatically by treatment with 0.61 mM
EDTA (Gibco). Passages 3–6
were used.
Cell studies.
The gradient region of the glass slide, including t
he PDMS well, was
separated from the rest of the slide by using a dia
mond pen and placed in a six-well
tissue culture plate. The plate was sterilized by u
ltraviolet light exposure for 5 min in a
laminar flow hood. HUVECs re-suspended in 5 mL seru
m-free EBM-2 containing 2%
BSA were added to the samples at a density of 10 00
0 cells /cm
2
. After 2 h, the
plates were gently washed twice with EBM-2 containi
ng 2% BSA, and imaged using
a 10x phase contrast objective on a Nikon Eclipse T
E 300 inverted microscope.
Images were captured on a Sony CCD color video came
ra (model DXC-151A)
equipped with Metamorph software. Fifteen images ta
ken randomly from 3 gradient
substrates were used to quantify cell attachment.
References
[1]
K. C. Zhang, M. R. Diehl, D. A. Tirrell,
J. Am. Chem. Soc.
2005
,
127
, 10136.
[2]
K. Kirshenbaum, I. S. Carrico, D. A. Tirrell,
ChemBioChem
2002
,
3
, 235.
[3]
N. L. Jeon, S. K. W. Dertinger, D. T. Chiu, I.
S. Choi, A. D. Stroock, G. M. Whitesides,
Langmuir
2000
,
16
, 8311.
[4]
X. Y. Jiang, Q. B. Xu, S. K. W. Dertinger, A. D
. Stroock, T. M. Fu, G. M. Whitesides,
Anal. Chem.
2005
,
77
, 2338, see Figure 7.