Incorporation of antifreeze proteins into polymer coatings using site-selective bioconjugation

Article abstract of DOI:10.1021/ja103038p

The diverse functional repertoire of proteins promises to yield new materials with unprecedented capabilities, so long as versatile chemical methods are available to introduce synthetic components at specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be cast into thin films. The polymer-protein conjugates retain the ability of the proteins to slow ice growth in subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials may prove useful as coatings for device components that must function at low temperature without ice buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using less protein, thus lowering the cost of these additives for biomedical applications.

Full text of DOI:10.1021/ja103038p

Published on Web 09/08/2010
Incorporation of Antifreeze Proteins into Polymer Coatings
Using Site-Selective Bioconjugation
Aaron P. Esser-Kahn,† Vivian Trang,† and Matthew B. Francis*,†,‡
Department of Chemistry, UniVersity of California, Berkeley, and Materials Sciences DiVision,
Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460
Received April 11, 2010; E-mail: francis@cchem.berkeley.edu
Abstract: The diverse functional repertoire of proteins promises to yield new materials with unprecedented
capabilities, so long as versatile chemical methods are available to introduce synthetic components at
specific sites on biomolecule surfaces. As a demonstration of this potential, we have used site-selective
strategies to attach antifreeze proteins found in Arctic fish and insects to polymer chains. This multivalent
arrangement increases the thermal hysteresis activity of the proteins and leads to materials that can be
cast into thin films. The polymer-protein conjugates retain the ability of the proteins to slow ice growth in
subzero water and can inhibit ice formation after attachment to glass surfaces. These inexpensive materials
may prove useful as coatings for device components that must function at low temperature without ice
buildup. The polymer attachment also allows higher thermal hysteresis values to be achieved while using
less protein, thus lowering the cost of these additives for biomedical applications.
Introduction
proteins (APFs, also known in the literature as “ice structuring”
proteins).3 Key residues of these sequences bind the surfaces
Protein-polymer hybrid materials afford compelling op-
portunities for blending the wide functional range of bio-
molecules with the convenient handling and processability
of polymers.1 The attachment of multiple proteins to each
polymer strand could enhance their collective behavior, and
polymer attachment could also lead to improvements in
solubility and stability. Recently, we reported a general
system for activation of proteins at the N- or C-terminus (or
both) to allow polymer attachment through oxime formation.2
The use of site-selective coupling chemistry improves the
homogeneity of the resulting material, making it significantly
more likely that the individual proteins will function in a
uniform manner. These methods are compatible with virtually
any desired sequence that can be expressed in Escherichia
coli, providing ready access to a diverse range of structural,
binding, and catalytic functionalities that can be imported
into the materials context.
of nascent ice crystals, preventing further growth at temperatures
within a characteristic thermal hysteresis range. These proteins
also inhibit the subsequent restructuring of small crystals into
expanded lattices.4 These properties are used to advantage in
organisms that live in subfreezing environments, slowing the
growth of ice crystal embryos that could otherwise cause tissue
damage. The ability of these proteins to depress the freezing
point of water and delay solid freezing has already been
exploited in the context of food additives,5 and their protective
properties are being explored for tissue cryopreservation, vaccine
storage, low-temperature surgeries, and the inhibition of gas
clathrate formation.6 We postulated that the attachment of these
proteins to polymer chains could provide two advantages over
the use of the proteins alone. First, multidomain oligomers of
antifreeze proteins have been shown to increase thermal
hysteresis beyond that of the single protein subunit,7 suggesting
that the connection of multiple copies of the protein to an
extended chain could display further enhancements in these
properties. Second, the attachment of polymer chains would
facilitate material handling and could serve as a convenient
means for preparing surface coatings that discourage ice
accumulation on critical machine components. Through the
development of a facile site-selective method to attach these
To continue the development of this strategy, we have
synthesized surface-attachable polymers that contain “antifreeze”
† University of California, Berkeley.
‡ Lawrence Berkeley National Laboratory.
(1) (a) Krishna, O. D.; Kiick, K. L. Pept. Sci. 2010, 94, 32–48. (b) Klok,
H. Macromolecules 2009, 42, 7990–8000. (c) Wang, P.; Sergeeva,
M. V.; Lim, L.; Dordick, J. S. Nat. Biotechnol. 1997, 15, 789–793.
(d) Christman, K. L.; Schopf, E.; Broyer, R. M.; Li, R. C.; Chen, Y.;
Maynard, H. D. J. Am. Chem. Soc. 2009, 131, 521–527. (e) Wang,
C.; Stewart, R. J.; KopeCek, J. Nature 1999, 397, 417–420. (f) Sui,
Z.; King, W.; Murphy, W. AdV. Mater. 2007, 19, 3377–3380. (g)
Murphy, W. L.; Dillmore, W.; Modica, J.; Mrksich, M. Angew. Chem.,
Int. Ed. 2007, 46, 3066–3069. (h) Christman, K. L.; Broyer, R. M.;
Tolstyka, Z. P.; Maynard, H. D. J. Mater. Chem. 2007, 17, 2021–
2027. (i) Christman, K. L.; Enriquez-Rios, V. D.; Maynard, H. D.
Soft Matter 2006, 2, 928–939.
(3) Davies, P.; Hew, C. FASEB J. 1990, 4, 2460–2468.
(4) Yeh, Y.; Feeney, R. E. Chem. ReV. 1996, 96, 601–618.
(5) Meldolesi, A. Nat. Biotechnol. 2009, 27, 682.
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13264 J. AM. CHEM. SOC. 2010, 132, 13264–13269
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Bioconjugation of Antifreeze Proteins in Polymer Coatings
A R T I C L E S
proteins to polymer backbones, we have found that both of these
aspects can be realized.
Results/Discussion
Two different AFPs were chosen for use in this study. The
first was a Type III antifreeze protein (T3AFP) from the ocean
pout fish, the structure, ice-binding site, and thermal hysteresis
of which have been characterized through careful mutagenesis
experiments.8 The second was a snow flea antifreeze protein
(SnAFP),9 which contains two disulfide bonds. Though exact
ice-binding residues of SnAFP remain unknown, it possesses
one of the highest thermal hysteresis values of any antifreeze
protein reported to date.
To allow attachment to the polymer chains, the proteins were
modified using expressed protein ligation (EPL)10 to install a
ketone at the C-terminus (yielding 3a, Figure 1a), as previously
reported.2 Following isolation of the protein, the utility of the
ketone group for polymer attachment was verified by exposing
the protein to a 2 kDa poly(ethylene glycol) (PEG-2k) chain
bearing an alkoxyamine group. As shown for T3AFP in Figure
1c, a gel shift was clearly discernible when the ketone was
present, indicating polymer attachment to yield 3c with over
90% yield. No shift was observed when protein 1c (which lacked
the ketone group) was exposed to the polymer alkoxyamine
under identical conditions. The analogous gel-shift assay could
not be performed for SnAFP, as in our hands it did not form a
well-defined band on the gel.11 Instead, MALDI-TOF MS
analysis was used to confirm modification following exposure
to benzyloxyamine alkoxyamine, Figure 1b. Oxime 3b was
observed as the sole product for both SnAFP and T3AFP,
confirming that both the EPL and the oximation steps had
proceeded with high conversion. In both cases the observed
molecular weights indicated that the N-terminal methionine
residues had been cleaved after protein expression, as would
be expected for proteins with alanine in the second position.12
To confirm the activity of the expressed proteins and verify
that the C-terminal ketone modification did not interfere with
their ice-binding properties, samples of T3AFP and SnAFP were
subjected to an ice-recrystallization inhibition assay.9 In this
experiment, capillary tubes containing 25 mg/mL samples of
protein (chosen for consistency with previous measurements
using these proteins9b) were flash frozen and visually recorded
as t ) 0 h. Each sample revealed uniform glassy ice that was
nonreflective, Figure 1d. The tubes were held at -6.0 °C for
4 h using a circulating cooling bath and a jacketed beaker. Visual
inspection was then used to determine if ice recrystallization
had occurred. In the tube containing 25 mg/mL bovine serum
albumin (BSA) as a control protein, significant restructuring of
the ice was observed, as indicated by the presence of reflective
crystalline surfaces. In contrast, the image of the ice in the tubes
containing the AFPs remained unchanged, indicating that the
Figure 1. Chemical synthesis of modified ice structuring proteins (or
antifreeze proteins, AFPs) for materials incorporation. (a) A native chemical
ligation strategy was used to modify the C-termini of AFPs with 2. The
resulting ketones were then coupled to alkoxyamines to yield stable oximes.
Conditions: (i) 250 mM BnONH2, pH 6.5, 1 h; (ii) 50 mM PEG-2k-ONH2,
pH 6.5, 16 h. The type III ice structuring protein (T3AFP) is depicted with
ice-binding residues highlighted in yellow. (b) T3AFP samples with (3a)
or lacking (1b) the ketone group were incubated with a 2 kDa PEG
alkoxyamine. Analysis by SDS-PAGE indicated a shift corresponding to
the formation of 3c only for the ketone-containing sample. (c) MALDI-
TOF analysis indicated complete modification of both T3AFP and SnAFP
with 2, followed by BnONH2. (d) An ice recrystallization inhibition assay
was used to confirm activity of the conjugates (each at 25 mg/mL) at -6.0
°C. Images were captured at t ) 0 and 4 h. Restructured ice crystals were
observed only in the BSA case (indicated by the red outline). Expanded
versions of these images have been included as Figure S4 in the Supporting
Information.
crystalline rearrangement had been inhibited. Similar behavior
was observed for modified T3AFP-3a, SnAFP-3a, and a sample
of T3AFP that was obtained from a commercial source, Figure
1d. Additionally, the circular dichroism (CD) spectrum of
SnAFP-3b was found to match previous reports (see Supporting
Information for details).9b Taken together, we interpret these
data to suggest that the protein expression and intein cleavage
protocols yield AFPs in the functional state and that the
C-terminal chemical modifications do not interfere appreciably
with their ice-binding activity.
Once the ice-binding properties had been confirmed, the
ability of the proteins to react with an alkyoxyamine-labeled
polymer was examined. A copolymer of 2-hydroxypropyl
methacrylamide (HPMA) and 3-aminooxypropyl methacryla-
mide was first prepared as previously described [MW )
175 000, PDI ) 1.75].2 A 50 mg/mL solution of this polymer
was then incubated with 20 mg/mL T3AFP-3a or SnAFP-3a at
pH 6.5 in 25 mM phosphate buffer for 2 h, Figure 2a. For
quantification purposes, some of the AFP samples were labeled
using the N-hydroxysuccinimide ester of tetramethylrhodamine
(TAMRA-NHS) before coupling to the polymers. Remaining
free protein (3a, MW ) 7000) was removed by three rounds
of ultrafiltration using a 100 kDa cutoff filter. UV-vis analysis
of the retained fraction indicated that 80-85% of the initial
(8) DeLuca, C.; Chao, H.; So¨nnichsen, F.; Sykes, B.; Davies, P. Biophys.
J. 1996, 71, 2346–2355.
(9) (a) Graham, L. A.; Davies, P. L. Science 2005, 310, 461. (b) Pentelute,
B. L.; Gates, Z. P.; Dashnau, J. L.; Vanderkooi, J. M.; Kent, S. B. H.
J. Am. Chem. Soc. 2008, 130, 9702–9707. (c) Tomczak, M. M.;
Marshall, C. B.; Gilbert, J. A.; Davies, P. L. Biochem. Biophys. Res.
Commun. 2003, 311, 1041–1046.
(10) Muir, T. W.; Sondhi, D.; Cole, P. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 6705–6710.
(11) Consistent with our own observations, there are no previous reports
of the SDS-PAGE analysis of SnAFP, despite several reports of
purification that yielded a single species via LCMS.
(12) Hirel, P. H.; Schmitter, M. J.; Dessen, P.; Fayat, G.; Blanquet, S. Proc.
Natl. Acad. Sci. U.S.A. 1989, 86, 8247–8251.
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The results of triplicate hysteresis experiments are tabulated
in Figure 2d. Compared to the monomeric ketone-labeled
proteins (3a) at identical concentrations, conjugation to the
polymer chains improved the thermal hysteresis value of T3AFP
by 60% at 1 mg/mL and doubled it for SnAFP at 0.5 mg/mL.
These are the largest hysteresis values reported for these
concentrations of protein. The addition of unconjugated poly-
mers had a negligible effect on the thermal hysteresis values
(Figure 2d) and did not appreciably change the properties of
the free monomeric proteins. We presume that the polymer-
conjugated AFPs can reach a higher local concentration on the
surface of the ice crystals, thus resulting in a thermal hysteresis
value that would be observed only at much higher concentrations
of monomeric protein.
The alkoxyamino groups on the polymer that remained after
protein attachment provide a convenient means for attaching
the material to surfaces bearing aldehyde or ketone groups. As
a test case, AFP-polymer conjugates were reacted with
commercially obtained glass slides that were coated with
aldehyde groups (Schott, Nexterion) at pH 6.5 for 2 h, Figure
3a. The samples were then rinsed with buffer for an additional
2 h to remove any unbound protein. Successful attachment was
verified by attaching TAMRA-labeled AFP-polymer conju-
gates, followed by fluorescence imaging, Figure 3b. Significant
fluorescence was observed only in cases in which the polymer
was present. Labeling and AFP incorporation were additionally
confirmed by observed differences in contact-angle measure-
ments for the glass surfaces, Figure 3c. Additionally, it was
possible to quantify the amount of AFPs that were attached to
the glass slides using bicinchoninic acid assays (see Supporting
Information). The concentration of the applied AFP-polymer
conjugates was not found to have a strong effect on the
properties of the coated slides, so a standard concentration of
50 mg/mL was used to prepare the surfaces in the experiments
that follow.
Figure 2. Polymer attachment for improved thermal hysteresis. (a) Ketone-
labeled AFPs (50 mg/mL) were attached to aminooxy-substituted HPMA
polymer 4 by oxime formation over a 3 h period. Only the type III ice
structuring protein (T3AFP) is depicted, with ice-binding residues high-
lighted in yellow. (b) To confirm attachment efficiency, TAMRA-labeled
protein was attached to 4 under the same conditions. Following concentration
of the solution using ultrafiltration (100 kDa cutoff), protein was retained
only in samples that displayed a ketone at the C-terminus. (c) Thermal
hysteresis was measured for the free proteins (1b or 3a) and the
protein-polymer conjugate (5). Ice crystals were grown at one end of the
tubes by the application of freeze spray. The samples were then subjected
to lowering temperatures at a rate of 0.01 °C/min. Thermal hysteresis was
determined by the onset of rapid ice crystal growth. (d) Table listing the
measured hysteresis values for the T3AFP and SnAFP derivatives.
To examine the antifrost properties of the coated surfaces, a
cooled steel surface was constructed (see Supporting Information
for a more thorough description). Samples were placed on a
surface cooled to -6.0 °C, which was the maximum temperature
found to form frost on the glass surface exposed to air at 20 (
1 °C and 40 ( 3% relative humidity. The glass samples were
then observed under a microscope, and the state of frost was
recorded every 15 min, Figure 3d. Initially moisture condensed
on each surface in small droplets, which slowly coalesced. As
would be expected, the morphology of the water drops closely
matched what had been seen in the contact angle measurements.
After 20 min, the aldehyde- and polymer-coated glass samples
had frozen and were continuing to gather frost. In contrast, the
glass slides coated with the T3AFP- and SnAFP-polymer
conjugates did not freeze after 30 min; instead, the accumulated
water droplets continued to expand. We presume that ice
nucleation occurs primarily at the cooled metal surface, and thus
the protein-polymer conjugates are in an ideal position to bind
the nascent crystals and inhibit their growth. After 45 min, ice
crystals were observed in the droplets, and the solutions were
completely frozen by the next time point (the 75 min result is
shown). Presumably, the bulk solution had cooled below its
freezing point after this amount of time, and the surface-confined
proteins could no longer reach the forming crystals. Thus, the
polymeric AFP coating is able to delay frost formation on cooled
surfaces, but it cannot prevent it entirely once sufficient water
has condensed with no access to the proteins. This material may
therefore prove most useful in applications where water ac-
ketone-bearing, TAMRA-labeled protein had been attached to
the polymer, Figure 2b. Protein samples lacking the ketone
groups (1c) but combined with the polymer under identical
conditions were retained only minimally by the concentrators.
On the basis of a calculation of relative concentrations and
percentage of attachment, we estimate 6-7 protein units per
polymer chain.
Polymer-AFP conjugates were next evaluated for their ability
to affect thermal hysteresis using a modified version of an assay
reported by De Vries.13 Thermal hysteresis is a difference in
the values of the freezing and thawing points of water. This
property results from the ability of AFPs to depress the solution
freezing point by binding growing ice crystals while not
significantly affecting the thawing temperature. Capillary tubes
were filled with 1 mg/mL solutions of the AFPs, and a small
(∼1 mm) ice crystal was formed at one end of the tube by
application of a freezing spray. The tubes were then placed in
a subzero solution, and the temperature was lowered at a rate
of 0.01 °C/min. The tubes were imaged using a digital camera
on a microscope every 5 min until rapid ice growth was
observed. The temperature at which this occurred was taken as
the hysteresis value, Figure 2c.
(13) DeVries, A. L. Methods Enzymol. 1986, 127, 293–303.
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A R T I C L E S
continue to explore the generality of this method for the efficient
synthesis of well-defined protein-containing materials.
Experimental Procedures
General Procedure for the Construction of the T3AFP-
pTXB1 and SnAFP-pTXB1 Expression Vectors. The T3AFP and
SnAFP E. coli optimized genes were received from Genscript in
pUC57 plasmids. The sequences for the genes were obtained from
the literature8,9 and are listed in the Supporting Information. An
alanine-glycine pair (AG) was added to the N-terminus to assist
in transamination reactions,14 and a lysine (K) was added to the C
terminus to assist in cleavage from the intein after expression.15
To allow molecular cloning, an NdeI restriction site was placed at
the N-terminal coding region of the gene, and a SapI site was
introduced at the C-terminal coding region. The gene was removed
from pUC57 using a double digestion with SapI and NdeI and was
ligated into the pTXB1 vector, which contained an ampicillin
resistance gene. The resulting plasmid was transformed into super-
competent NEB 5R cells (NEB) using electroporation. The cells
were then plated and grown overnight, and colonies were selected
by virtue of ampicillin resistance. The presence of the AFP coding
segment was verified in the resulting plasmids by both restriction
digestion and sequencing.
Small-Scale AFP Expression. T3AFP-pTXB1 or SnAFP-
pTXB1 plasmids were transformed into T7 express LysY/Iq
competent E. coli (NEB) via electroporation utilizing a micro-pulser
(Biorad, Hercules, CA) and spread onto Luria broth (LB) agar plates
with ampicillin at 100 µg/mL. Cells from selected colonies were
grown in 1 L of LB-containing ampicillin at 100 µg/mL at 37 °C
until an optical density (OD) of 0.7 was observed at 600 nm. Protein
expression was then induced by the addition of 1 mL of 0.3 mM
isopropyl ꢀ-D-thiogalactopyranoside (IPTG). In the case of T3AFP,
the broth cultures were grown for an additional 16 h at 16 °C. For
SnAFP, the broth cultures were grown for 4 h at 30 °C after
induction. For each sample, the cells were collected via centrifuga-
tion for 20 min at 8000 rcf at 4 °C. Protein expression was verified
by the presence of the desired AFP-intein-chitin fusion by sodium
dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE).
The cells were then resuspended in 10 mL of lysis buffer (0.02 M
Tris, 0.15 M NaCl, 5 mM EDTA, pH 8.0) by vortexing. The cells
were lysed by sonication using a Branson digital sonifier (VWR
Scientific, West Chester, PA) for 20 min with a blunt-ended tip.
Debris was removed by centrifugation at 8000g for 15 min to give
a light brown solution.
Large-Scale AFP Expression. T3AFP-pTXB1 or SnAFP-
pTXB1 plasmids were transformed into T7 express competent E.
coli via electroporation utilizing a micro-pulser and plated on LB
agar plates with ampicillin at 100 µg/mL. Cells from selected
colonies were grown overnight in 4 L of LB containing ampicillin
at 100 µg/mL. This 4 L was then introduced into 120 L of LB
containing ampicillin at 100 µg/mL in a 150 L BioFlo Pro industrial
fermentor (New Brunswick Scientific Co. Inc., Edison, NJ). Cells
were grown at 37 °C until an OD600 of 0.6 was reached, at which
point IPTG was added to a final concentration of 300 µM. For
T3AFP, the temperature was lowered to 16 °C, and the cells were
allowed to grow for an additional 18 h. For SnAFP, the temperature
was lowered to 30 °C, and the cells were allowed to grow for 4 h
following induction. For all samples, the cells were isolated using
a Sharples super-centrifuge over the period of 1 h. The cells were
scraped from the sides of the centrifuge and heat-sealed in plastic
envelopes. The samples were then frozen for storage and transport
between two blocks of dry ice. To obtain protein, portions of the
cells were thawed and lysed by homogenization using an Emulsi-
flex-C3 homogenizer (Avestin Inc., Ottawa, Canada) for three
Figure 3. Preparation and evaluation of freeze-resistant surfaces. (a) Free
alkoxyamine groups remaining after protein attachment were coupled to
aldehyde groups on glass slides (conditions: pH 6.5, 3 h, 50 mg/mL protein).
Only the type III ice structuring protein (T3AFP) is depicted, with ice-
binding residues highlighted in yellow. (b) To confirm binding, free proteins
and protein-HMPA conjugates were labeled with TAMRA. Successful slide
attachment was accompanied by red fluorescence. (c) Contact angle
measurements were also used to confirm polymer attachment. (d) Coated
glass slides were cooled to -6.0 °C at controlled humidity. Condensed
water froze significantly faster on samples that lacked the proteins. Images
were collected at 40× magnification.
cumulation on a surface is not as pronounced, such as natural-
gas pipelines and wind turbines.
Conclusion
These new antifreeze-protein conjugates have demonstrated
that protein function can easily be imported for different
purposes and onto different surfaces using site-specific oxime
formation. Simultaneously, it has been shown that the properties
of a protein can be enhanced by the attachment of polymers.
Because a specific face of the AFPs binds the growing ice
crystals, it is likely advantageous to have a site-selective
attachment strategy. It should be possible in the future to test
these materials for inhibition of frost and ice formation in a
number of different situations. As one possibility, recent work
has shown that AFPs can inhibit the formation of gas clathrates,8
suggesting that these coatings may find use in the natural-gas
and energy industries. In parallel to these studies, we will
(14) Scheck, R. A.; Dedeo, M. T.; Iavarone, A. T.; Francis, M. B. J. Am.
Chem. Soc. 2008, 130, 11762–11770.
(15) IMPACT-CN Instruction Manual; New England Biolabs: Ipswich, MA,
2006; http://www.neb.com/nebecomm/products/productE6900.asp.
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rounds. Debris was removed by centrifugation at 8000g for 15 min
to give a murky brown solution. Typical expression yielded 500 g
of wet cell mass per 120 L of broth.
25 µL microcapillary tubes (Drummond Scientific Co.). Each end
was then flame-sealed, leaving at least a 3 cm “air gap” between
sample and flamed end. A single ice crystal measuring less than
0.5 cm was then created at the left side of the microcapillary tube
by the application of freeze spray (Innovera, Deerfield, IL). Each
sample was placed in a jacketed bath that was cooled to -0.2 °C
with chilled isooctane. The bath was illuminated from below with
a light source that passed through a piece of polarizing film before
reaching the samples (polarizing film sheet 30 cm × 30 cm, Arbor
Scientific, Ann Arbor, MI). The temperature of the bath containing
the samples was lowered at a rate of 0.01 °C/min, and images were
captured every 5 min. A second piece of polarizing film was placed
between the top of the bath and the lens of the microscope at a 90°
angle to the first filter, such that light was unable to pass through
the filters unless its polarization had been altered by ice crystals.
Images were captured every 10 min with the second polarizing filter
in place.
Confirmation of Polymer Attachment Using Ultracentrifu-
gation. Samples that were 100 µM in T3AFP or SnAFP were
reacted with 500 µM TAMRA-NHS (Invitrogen, Carlsbad, CA) in
0.1 M sodium phosphate, pH 8.0, containing 1 mM EDTA. The
reaction proceeded for 3 h and was quenched by elution through a
NAP-5 column (GE Healthcare) to remove unreacted TAMRA-
NHS. The eluted protein (in 50 mM ammonium carbonate (AC)
buffer, pH 6.5) was then concentrated to 50 mg/mL for T3AFP
and 25 mg/mL for SnAFP. A 50 µL aliquot was reacted with
alkoxyamino-substituted HPMA polymer in a manner identical to
that described above. A fluorescence emission spectrum was
obtained from 550 to 700 nm (the integration over this wavelength
range was recorded as I) from 1 mL of sample. Samples were then
subjected to three rounds of ultrafiltration (100K MWCO, Milli-
pore). The retentate was collected and brought to a volume of 500
µL with AC buffer, and then the same fluorescence measurement
was performed (yielding an integrated spectral value of R). The
percent retention was calculated by the equation (I - R)/I × 100
and is graphed in Figure 2b. The error bars represent (1 standard
deviation from the mean for three independent measurements.
Attachment of Polymer-Protein Conjugates to Aldehyde-
Coated Glass Slides. Lyophilized samples of SnAFP or T3AFP
were weighed on a microbalance, with a typical weight of ∼2 mg,
and dissolved in PBS (10 mM sodium phosphate, 100 mM NaCl,
pH 6.5) to a concentration of 50, 25, 10, or 0 mg/mL. The solution
was then reacted by the addition of 5 mg aliquots of alkoxyamino-
substituted HPMA polymer from a stock solution of 100 mg/mL.
The samples were allowed to react for 1 h at room temperature.
The solution was diluted to 600 µL and then pipetted onto a cut
piece of aldehyde-coated glass (Nexterion Slide AL, Schott-
Nexterion, Jena, Germany) typically measuring 0.983 × 0.350 in.
The solution covered the entire surface. The glass had been
previously cleaned by sonication in H2O for 30 min. The glass
sample was placed in a micro Petri dish (Millipore) that was then
sealed with parafilm. The reaction on the glass surface was allowed
to proceed for 2 h at room temperature. The liquid containing the
remaining polymer and protein was then removed via pipet, and
the glass was washed first in PBS for 30 min and then with H2O
for 30 min by rotary shaking. The glass was air-dried and
subsequently used for the frosting inhibition experiments.
Affinity Purification of SnAFP and T3AFP and Modifica-
tion of the C-Termini through Native Chemical Ligation. The
methods used to produce chemically modified SnAFP and T3ASP
were based on a literature protocol.2a The same procedure was used
for both proteins. Before use, all buffers were cooled to 4 °C. The
SnAFP- or T3AFP-containing lysate was washed over 200 mL of
chitin resin (New England BioLabs, Inc., Ipswich, MA) in a 500
mL centrifuge flask for 1 h. The resin was then isolated by
centrifugation at 4 °C. The resulting resin-bound protein was placed
in a 500 mL Steritop filter unit (Millipore, Billerica, MA) and
washed with 4 L of wash buffer (0.02 M Tris, 0.5 M NaCl, 1 mM
EDTA, pH 7.5) that was cooled to 4 °C. Binding to the column
was confirmed by SDS-PAGE analysis of the eluent. A 150 mL
solution of 50 mM MESNa and 5 mM cysteine-ketone in wash
buffer (pH adjusted to 7.5) was flowed over the resin-bound protein
using suction. The column bed was then allowed to stand in a
minimal amount of this solution at 4 °C for 15 h with protection
from light. The protein was eluted from the column with the addition
of 200 mL of wash buffer. Purified protein was then buffer-washed
three times with wash buffer using Amicon Ultra 15 mL 3000
MWCO (Millipore) and incubated with an additional 25 mL of
chitin resin for 1 h. The chitin resin was removed by filtration
through a Steriflip filter unit (Millipore). Purified protein was then
buffer-exchanged into 25 mM sodium phosphate buffer (pH 6.5)
using Amicon Ultra 15 mL 3000 MWCO centrifugal ultrafiltration
membranes. The resulting solution was then passed through two
sequential PD-10 (GE Healthcare) buffer exchange columns to
transfer the protein into 50 mM ammonium carbonate buffer (pH
7.9) at 4 °C. The resulting solution was snap-frozen using liquid
N2 and lyophilized to produce a dried powder that was stored at
-20 °C. For all further reactions, the powder was first dissolved
in the designated buffer. This purification typically yielded 1 mg/L
of purified, modified protein. The entire procedure, except for
lyophilization, was performed in a cold room maintained at 4 °C.
Protein identity and purity were evaluated by SDS-PAGE with
Coomassie staining and/or by using MALDI-TOF MS.
Ice Recrystallization Inhibition Assay. The procedure was
based on the methods of Kent et al.9b and Knight et al.16
Lyophilized samples were weighed using a microbalance and
dissolved in phosphate-buffered saline (PBS, 10 mM sodium
phosphate, 100 mM NaCl, pH 7.5). Samples were then loaded by
capillary action into 25 µL microcapillary tubes (Drummond
Scientific Co., Broomall, PA), and each end was flame-sealed while
leaving at least a 1 cm “air gap” between sample and flamed end.
Samples were then snap-frozen for 10 s in 2,2,4-trimethylpentane
(isooctane) cooled with dry ice and immediately placed in a jacketed
beaker cooled to -6.0 °C using the same solvent. Images were
taken at 40× total magnification with lighting from below.
Authentic antifreeze protein type III (T3AFP, A/F Protein, Waltham,
MA) was used as a positive control. Bovine serum albumin (BSA,
Aldrich, St. Louis, MO) was used as a negative control.
Attachment of Polymers to Proteins for Thermal Hysteresis
Experiments. Samples of SnAFP and T3AFP were weighed on a
microbalance. The proteins were dissolved in 25 mM ammonium
carbonate buffer (pH 6.5) at a concentration of 50 mg/mL for
T3AFP and 25 mg/mL for SnAFP. The lower concentration was
chosen for SnAFP so that measurements of SnAFP and T3AFP
could be performed over the same temperature range. Alkoxyamine-
substituted HPMA polymer was then added to the solution at a
concentration of 25 mg/mL from a stock solution of 50 mg/mL in
the same buffer. The two components were allowed to react for
2 h at room temperature. The reaction solution was then diluted to
1/50th the initial concentration and loaded via capillary action into
Confirmation of Polymer Attachment Using Fluorescence.
Samples of T3AFP or SnAFP were prepared at 100 µM in 0.1 M
sodium phosphate buffer, pH 8.0, containing 1 mM EDTA.
TAMRA-NHS (Invitrogen) was added to a concentration of 500
µM. After 3 h, the reaction was quenched by elution through a
NAP-5 column (GE Healthcare) to remove unreacted TAMRA-
NHS. The eluted protein (in PBS) was then concentrated to 50 mg/
mL for both T3AFP and SnAFP in a volume of 40 µL, reacted
with alkoxyamino-substituted HPMA polymer, and subsequently
pipetted onto aldehyde-coated glass as noted above. All samples
designated “3a” in Figure 3b were incubated with T3AFP, SnAFP,
or rhodamine-labeled ketone S1 (see Supporting Information) while
(16) Knight, C. A.; Wen, D.; Laursen, R. A. Cryobiology 1995, 32, 23–
34.
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Bioconjugation of Antifreeze Proteins in Polymer Coatings
A R T I C L E S
excluding polymer 4. They were then subjected to identical washing
and sonication conditions. Fluorescent measurements were obtained
using a Typhoon 9410 (GE Healthcare) flat bed fluorescent scanner.
The emission filter was set at 588-Cy3,TAMRA using a green laser
(532 nm). Images were false colored (red) and identically contrast/
brightness adjusted using ImageQuant software.
Procedure for Frosting Inhibition Experiments. All experi-
ments were conducted on a steel surface that was cooled from below
using a circulating bath (Neslab RTE-111). The surface was first
cooled to -6.0 °C. The atmosphere surrounding the surface was at
ambient laboratory room temperature (20 ( 1 °C), pressure, and
relative humidity (40 ( 3%). Protein-modified glass slides were
removed from the washing solution, dried, and then placed on the
steel plate. Images were captured every 5 min for the first 30 min
and every 15 min thereafter. All images are reported at 40× total
zoom. The general trend for each plate was to accumulate water
condensation and then freeze after a certain amount of time. The
T3AFP- and SnAFP-coated plates were observed to resist ice
formation until a substantial amount of water had accumulated over
the surface (see Figure 3d).
Acknowledgment. This work was supported by the NSF
(0449772) and the Berkeley Chemical Biology Graduate Program
(NRSA Training Grant 1 T32 GMO66698). We thank Crystal Chan
and the Berkeley Fermentation Facility, Virginia K. Walker, and
Troy A. Moore for helpful discussions.
Supporting Information Available: Supporting Figures S1-S4
and general experimental details. This material is available free
of charge via the Internet at http://pubs.acs.org.
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