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A Recombinant Triblock Protein Polymer With Dispersant and Binding

A Properties
Recombinant for Triblock Protein Polymer With Dispersant and Binding
Digital Printing
Properties for Digital Printing
Min Qi, John P. O’Brien, Jianjun Yang
DuPont Central Research and Development, Experimental Station, P. O. Box 80402, Wilmington, DE 19880-0402

Received 21 May 2007; revised 12 October 2007; accepted 22 October 2007

Published online 30 October 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20878

ABSTRACT: digital printing inks was demonstrated. # 2007 Wiley

A structured triblock protein was designed to explore the Periodicals, Inc. Biopolymers (Pept Sci) 90: 28–36, 2008.

potential of engineered peptides to function as high- Keywords: phage display; pigment-binding peptide; cellu-

performance ink dispersants and binders. The protein lose-binding peptide; dispersant

consists of three functional elements, including a pigment

This article was originally published online as an accepted
binding domain, a hydrophilic linker, and a printing preprint. The ‘‘Published Online’’ date corresponds to the
surface binding domain. To construct such a chimeric preprint version. You can request a copy of the preprint by
protein, a carbon black binding peptide, FHENWPS, and emailing the Biopolymers editorial office at biopolymers@wiley.
a cellulose binding peptide, THKTSTQRLLAA, were
identified from phage display libraries through
biopanning, based on their strong and specific binding INTRODUCTION

olymeric dispersants are widely used to stabilize pig-
affinities to carbon black and cellulose. They were used as
ments in coating systems such as paints and finishes,
carbon black and cellulose binding domains, respectively, and in inkjet printing inks.1–3 The dispersant serves
in a recombinant triblock protein. A linker sequence, to form a shell around the pigment particle, prevent-
PTPTPTPTPTPTPTPTPTPTPTP, was adapted from ing flocculation and coagulation. In aqueous sys-
tems, the pigment dispersion is generally stabilized using
endoglucanase A of the bacterium Cellulomonas fimi, as
nonionic or ionic techniques. The nonionic technique stabil-
a small, rigid, and hydrophilic interdomain linker. When izes the pigment by a polymer that has a hydrophilic region
incorporated into the triblock structure between the extending into the aqueous phase to provide entropic or
carbon black and cellulose binding sequences, the linker steric stabilization. It is not sensitive to pH changes or ionic
sufficiently isolates these two elements and allows dual contamination but is sensitive to water. If used in ink appli-
cations, the pigment will tend to smear upon exposure to
binding activity. The structured triblock protein was
moisture. In the ionic technique, the pigment particles are
shown to disperse carbon black particles and attach it to stabilized by a polymer consisting of ionically charged mono-
paper surfaces. Thus, the utility of structured proteins mers. The polymer provides stabilization through a charged
having useful dispersant and binding properties for double-layer mechanism, whereby ionic repulsion hinders
the particles from flocculation. Since the neutralizing com-
ponent tends to evaporate after application, the polymer has
reduced water solubility and the final product is not water-
sensitive. Structured polymeric dispersants, such as block
Correspondence to: Jianjun Yang; e-mail: jianjun-gene.yang@usa.dupont.com
and graft polymers, provide both steric and ionic stabiliza-
tion, and thus make the most robust pigment dispersions.1
Proteins have previously been used as dispersants. For
C 2007 Wiley Periodicals, Inc. example, chemically modified casein, collagen, albumin, and

28 PeptideScience Volume 90 / Number 1

Recombinant Triblock Protein Dispersant 29

gelatin have been applied as dispersants in color formula- Tris-HCl, pH 9.1. The eluted phages were titered, sequenced, and
tions.4 Polyanionic peptides such as polyaspartate have also amplified as described by the supplier. These phage particles were
then subjected to the next run of biopanning. Four iterations were
been used to disperse inorganic mineral particles.5 However,
carried out with 0.1%, 0.5%, 1%, and 2% Tween-20 in TTBS-X,
these sequences have not been specifically designed to utilize sequentially.
the full potential of protein-based dispersants. Their func-
tions mainly depend on natural properties of the proteins,
and thus do not meet the needs of sophisticated ink and GST-Peptide Constructs and
coating formulations. Recombinant DNA methods allow for Alanine-Scanning Mutagenesis
the design of protein polymers that have highly specific pri- A master plasmid of pGSTf was derived from Gateway pDEST15
(Invitrogen, Carsbad, CA), by removing a fragment of unnecessary
mary sequences and functional domains. Hence, it should be nucleotides and adding unique NcoI, SacI, NotI, and AscI sites at 30
possible to create a protein-based dispersant that offers supe- end of the GST coding sequence just before its stop codon. To link a
rior functionality over classic chemical-based dispersants. In binding peptide or its alanine-scanning mutant to the C-terminal of
this study, a triblock protein was designed and constructed as GST, two oligonucleotides were synthesized. One had the sequence
a pigment dispersant for digital printing. The first block con- GGCCGCCX(n)TAAGG, and other was CGCGCCTTAY(n)GGCGGC
CGC. X(n) and Y(n) represented sense and antisense strands of the
sists of a pigment binding peptide that offers strong and spe-
peptide coding sequence. The oligonucleotides were annealed to
cific binding affinity to the surface of the pigment particle. form a double-stranded DNA fragment, encoding for the peptide. It
The second block is a hydrophilic linker that not only isolates also had 50 and 30 sticky ends that were complementary to SacI and
the affinity blocks but also stabilizes pigment dispersion AscI sites, respectively. Therefore, it could be inserted to down-
through direct interaction with aqueous media. The last stream of GST coding sequence between SacI and AscI sites in
pGSTf. The resulting construct encoded for the GST-peptide fusion,
block is a cellulose binding peptide that tightly binds to cel-
lulose surfaces, such as paper and fabric. Peptide sequences
for the substrate binding blocks were obtained by biopanning
of peptide display libraries. The hydrophilic linkers were GST-TBP Constructs
selected from known natural linker sequences. Such a tri- Five complementary and overlapped oligonucleotides were synthe-
block architecture structure was targeted to function as a dis- sized according to the coding sequence of IEGR(CB72::ST37::
CEL121), and assembled by annealing. The resultant double-
persant/binder, offering improved dispersion stability and
stranded DNA was amplified by PCR and cloned into Gateway
image durability for digital printing. pENTR/D-TOPO (Invitrogen). The resultant pENTR-TBP101
encoded for IEGR(CB72::ST37::CEL121). The ST37 coding region
has an NgoMI site at the beginning and a KasI site at the end. It was
replaced by a synthetic PT23 coding region through unique NgoM
MATERIALS AND METHODS I/Kas I digestion, forming pENTR-TBP201. The TBP structures in
pENTR-TBP101 and pENTR-TBP201 were constructed into Gate-
Biopanning With a Phage Display Library way pDEST17 and pDEST15 (Invitrogen) by taking advantage of
Ph.D.-7 and Ph.D.-12 phage display libraries were purchased from the site-specific recombination of bacteriophage lamda, as described
New England BioLabs (Beverly, MA). Carbon Black FW-18 by the manufacturer, which generated pINK101, pINK201,
(Degussa, Piscataway, NJ) and long fibrous cellulose (Sigma, St pINK151, and pINK251. Similarly, the PT23 coding region in
Louis, MO) were used as binding substrates. Biopanning was carried pENTR-TBP201 was further replaced by shortened PT coding
out using methods provided by New England BioLabs. Since the sequences, and then incorporated into pDEST15, resulting in
binding substrates were solids, the procedure was modified slightly. pINK2a51, pINK2b51, and pINK2c51.
During the panning, an appropriate amount of solid substrate was
placed in a microcentrifuge tube and filled with a blocking buffer
consisting of 5 mg/mL bovine serum albumin (BSA) and 0.1M Expression and Purification of Fusion Proteins
NaHCO3 at pH 8.6. The substrate was incubated for 1 h at 48C and Expression plasmids were transformed into BL21 E. coli cells.
washed six times with TTBS-0.1%, a TBS buffer (150 mM NaCl, Expression of fusion proteins was induced by L-arabinose for 4 h at
50 mM Tris-HCl, pH 7.5) containing 0.1% Tween 20. The substrate 378C and examined by NuPAGE SDS-PAGE (Invitrogen) with Coo-
was then incubated with 1 mL of TTBS-0.1% containing 2 3 1011 massie Blue staining. To purify the insoluble 6xHis-TBP fusion pro-
phage (10 lL of original library) and 1 mg/mL BSA for 10–20 min teins, E. coli cells collected from 50 mL culture were treated in
at room temperature. After this time, the substrate was washed five 1.5 mL CelLytic B bacterial cell lysis extraction reagent (Sigma) con-
times with TTBS-X (TBS with a designated amount of Tween 20, as taining 5 lg DNase I and 30 lL proteinase inhibitor cocktail
given infra for the specific experiments) containing 1 mg/mL BSA (Sigma), for 30 min at room temperature. The insoluble matter was
and then washed an additional five times with TTBS-X alone. Sub- dissolved in 5 mL of 8M urea. The fusion proteins were purified
strate-binding phage were eluted by incubating the substrate with under denaturing conditions through a 0.5 mL Ni-NTA column
1 mL of elution solution (1 mg/mL BSA, 0.2M glycine-HCl, pH 2.2) (Qiagen, Valencia, CA), following a protocol provided by Qiagen.
for 7 min and neutralized immediately by adding 150 lL of 1M Fusion protein eluted from the column was dialyzed against IEB

Biopolymers (Peptide Science) DOI 10.1002/bip

30 Qi, O’Brien, and Yang

buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 0.1 for each sample, and the average OD405 values and standard devia-
mM EDTA, and 5% glycerol). The dialysis buffer contained 8M urea tions were calculated accordingly.
initially. The urea concentration was gradually reduced to zero dur-
ing the course of dialysis to bring the fusion protein back to a water-
soluble form. To purify the soluble GST-TBP fusion protein, E. coli Microscale Dispersion and Attachment Assays
lysate was loaded on to a 1.5 mL glutathione-agarose column For the dispersion assay, a sample containing 50 lg carbon black
(Sigma). The column was washed with TBS buffer containing 1% FW-18 and 0.6 nmol TBP in 10 lL IEB buffer was used. After gentle
Tween-20, and the fusion protein was eluted with FST elution buffer mixing, 5 lL of the sample was dropped onto a glass slide (VWR
(50 mM Tris-HCl, pH 9.5, 10 mM reduced glutathione). To quickly Scientific, Media, PA) and observed for pigment dispersion after
purify GST-peptide fusion proteins, a B-PER GST fusion protein pu- standing for 2 min. In the attachment assay, 50 lg carbon black
rification kit (Pierce, Rockford, IL) was used. The purified GST-TBP FW-18 was suspended in 10 lL IEB buffer. Then, 5 lL IEB buffer
and GST-peptide fusion proteins were dialyzed against IEB buffer. In containing 0.6 nmol TBP201 was added to the suspension and
all protein purification, NuPAGE SDS-PAGE was employed to check gently mixed for 20 min. Five microliters of sample was then spot-
protein quality, and the Bio-Rad protein assay reagent (Bio-Rad, ted on 0.8 3 8 cm Whatman 5 filter paper (Whatman, Clifton, NJ).
Hercules, CA) was used to determine protein concentrations. The paper was washed for 2 min with 10 mL TTBS-0.1%. Pigment
retained on the paper before and after washing was evaluated. For
the competition tests in both dispersion and attachment assays, free
Factor Xa Treatment CB72 and CEL121 peptides were purchased from Synpep (Dublin,
A 4.5-mL GST-TBP201 solution, containing 10 mg GST-TBP201, CA) and used as described later.
was treated with 100 lg biotin-labeled restriction protease factor Xa
(Roche, Mannheim, Germany) for 18 h at 48C to release TBP201
from GST. In order to remove factor Xa, the reaction was mixed RESULTS
with streptavidin suspension (Roche) for 1 h at 48C and centrifuged
at 4000 rpm for 5 min at 48C. The Xa-free supernatant was then
passed through a 1 mL immobilized glutathione column (Pierce) at Identification and Characterization of Carbon Black
48C to remove GST-containing proteins. Finally, the eluent was fil- Binding Peptides and Cellulose Binding Peptides
tered through a Centrocon YM-10 (Millipore, Bedford, MA) by cen- A triblock protein dispersant as described earlier contains a pig-
trifuge at 48C, resulting in ~4.5 mL TBP201 solution. ment-binding peptide and a cellulose-binding peptide. Phage
display biopanning was employed to identify affinity peptides
ELISA Binding Assay from 7- and 12-mer peptide libraries, following a protocol
A phage-ELISA binding assay was carried out in a MultiScreen-HV briefly described in the Materials and Methods section. Biopan-
(0.45 lM) 96-well filtration plate (Millipore). Carbon black FW-18 ning was conducted against 250 lg of carbon black and long
or Sigmacell cellulose (type 20, Sigma) was placed in the wells, fibrous cellulose, respectively. DNA samples from 25-phage
blocked by 200 lL of blocking buffer (5 mg/mL BSA, 0.1M plaques were sequenced in each run to identify the actual dis-
NaHCO3, pH 8.6) for 1 h, and then washed five times using 200 lL
played peptide sequences. Based on the results, sequences that
TTBS-0.1% buffer (TBS buffer containing 0.1% Tween 20). Approx-
imately 8 3 1010 phage particles and 0.2 mg BSA were mixed with consistently interacted with carbon black or cellulose and were
200 lL TTBS-0.1% and added to each well to interact with the sub- continuously enriched after each run were selected. These selec-
strate for 15 min. The wells were washed four times using 200 lL tions included 7-amino acid carbon black binding peptides
TTBS-0.5% containing 0.2 mg BSA, and four more times using 200 lL (CB71 and CB72), 12-amino acid carbon black binding pepti-
TTBS-0.5%. An M13-HRP conjugate (Amersham Bioscience, Pis- des (CB121 and CB122), 7-amino acid cellulose binding pepti-
cataway, NJ) was diluted 5000-fold with TBS containing 1 mg/mL
BSA and added to each well to react with the substrate-bound phage
des (CEL71, CEL72, and CEL73), and 12-amino acid cellulose
particles for 1 h. The wells were washed five times with 200 lL binding peptides (CEL121, CEL122, and CEL123). Amino acid
TTBS-0.1%. Finally, 200 lL of ABTS solution (0.4 mM 2,20 -azino- sequences of these peptides are given in Table I.
bis(3-ethylbenzthiazoline-6-sulfonic acid), 0.05% H202, 50 mM In biopanning, a peptide sequence may be enriched for
sodium citrate, pH 4.0) was added to each well to react for 25 min, reasons other than substrate-specific binding activity. For
resulting in a colored solution. In the assay, solution changes in
example, fast growth of a particular phage may occur during
each step were carried out using a vacuum manifold. The final
colored solution in the filtration plate was transferred into a regular amplification and may render a false positive. CB72 and
96-well plate and OD405 was measured on a Victor-3 1420 Multiple CEL73 were suspected of this behavior, since they were
Label Counter (Perkin Elmer, Shelton, CT). GST-ELISA and 6xHis- selected against carbon black and cellulose, respectively, but
ELISA binding assays were carried out, following a procedure simi- shared the same sequence. To avoid false positives, we con-
lar to the procedure mentioned earlier. In the GST-ELISA binding
firmed substrate-specific binding activity by using a Phage-
assay, 0.1 nmol GST fusion protein and 2500-fold diluted GST-HRP
conjugate (Sigma) were used. In the 6xHis-ELISA binding assay, 0.1 ELISA binding assay. In the assay, 8 3 1010 copies of phage
nmol 6xHis fusion protein and 5000-fold diluted Penta-His HRP carrying CB71, CB72, CB121, and CB122 were exposed to
conjugate (Qiagen) were used. Triplicate reactions were conducted 75 lg of carbon black FW-18 to measure carbon black bind-

Biopolymers (Peptide Science) DOI 10.1002/bip

Recombinant Triblock Protein Dispersant 31

Table I Phage-ELISA Binding Assay of Selected alanine-replacement mutants (i.e., GST-CB72A1 to GST-
Affinity Peptides CB72A7); 10 GST-CEL121 alanine-repalcement mutants
(i.e., GST-CEL121A1 to GST-CEL121A10); and 2 GST-
Peptide Sequence OD405a
CEL121 glycine-replacement mutants (i.e., GST-CEL121G11
Carbon black binding and GST-CEL121G12). GST and its fusion partners were
CB71 MPPPLMQ 2.200 6 0.120 connected by the short peptide, SAAA. These fusion proteins
CB72 FHENWPS 3.630 6 0.055 are listed in Table II. They were produced, purified, and
Ctrl71 SNRDLVY 0.079 6 0.035 tested using the GST-ELISA binding assay. In the GST-
CB121 RTAPTTPLLLSL 0.552 6 0.030
ELISA binding assay, 0.1 nmol GST-peptide protein was
CB122 WHLSWSPVPLPT 2.421 6 0.236
Ctrl121 SSNLNLSWVQDT 0.112 6 0.011 exposed to 37.5 lg of carbon black to measure carbon black
binding activity and to 600 lg of Sigmacell cellulose to mea-
Cellulose binding
sure cellulose binding activity. Binding activity was then
CEL71 VPRVTSI 0.157 6 0.027
CEL72 MANHNLS 0.221 6 0.010 determined by ELISA against GST and quantified via OD405
CEL73 FHENWPS 0.071 6 0.011 measurements (Table II). A comparison between GST-CB72
Ctrl71 SNRDLVY 0.062 6 0.007 and its mutants indicated that most residues contribute to
CEL121 THKTSTQRLLAA 0.406 6 0.020 the carbon black binding activity of CB72. However, phenyl-
CEL122 KCCYVNVGSVFS 0.299 6 0.013
alanine (F1), histidine (H2), and tryptophan (W5) played
CEL123 AHMQFRTSLTPH 0.086 6 0.010
Ctrl121 SSNLNLSWVQDT 0.096 6 0.014 more critical roles. Their replacement by alanine had signifi-
cantly reduced the activity by 22%, 19%, and 32%, respec-
An average of triplicate assays. tively. Since all three amino acids have an aromatic side
chain, it suggested that hydrophobic interactions might play
ing activity, while the same amount of phage carrying a role in the carbon black binding activity of CB72. Simi-
CEL71, CEL72, CEL73, CEL121, CEL122, and CEL123 were larly, a comparison between GST-CEL121 and its mutants
exposed to 150 lg of Sigmacell cellulose to measure cellulose showed that, although every amino acid residue in CEL121
binding activity. The amount of phage specifically bound contributes to cellulose binding activity, critical residues
with substrate was then determined by ELISA analysis were lysine (K3), glutamine (Q7), arginine (R8), and leucine
against M13 phage and quantified through optical density (L9 and L10). Alanine-substitution of any of these residues
(OD405) measurements. Two phages obtained from the resulted in a more than 20% reduction of cellulose binding
Ph.D.-7 and Ph.D.-12 libraries served as negative controls, activity. Because lysine, glutamine, and arginine contain po-
respectively. One phage contained the randomly selected 7- lar side chains that render them highly hydrophilic, cellulose
amino acid sequence SNRDLVY, and was designated Ctrl-71. binding activity appears to be different from that of CB72,
The other control contained the 12-amino acid sequence which is controlled primarily by hydrophobic interactions.
SSNLNLSWVQDT, and designated Ctrl-121. After compar-
ing ELISA data to control phages, shown in Table I, we con- Triblock Proteins Exhibit Dual Binding Activity
cluded that CB71, CB72, CB121, and CB122 are carbon Protein-based dispersants/binders should ideally exhibit both
black binding peptides while CEL71, CEL72, CEL121, and pigment and print surface binding activity. To reconstitute
CEL122 are cellulose binding peptides. CEL73 and CEL123 such dual binding functionality, peptides CB72 and CEL121
did not show specific binding activity to cellulose fibers, and were chosen as the carbon black and cellulose binding ele-
were eliminated from further investigation. ments, respectively. Two linker sequences adapted from natu-
The top-ranked binding peptides, CB72 and CEL121, ral enzymes were used to connect two affinity sequences.
were further characterized by alanine-scanning mutagenesis, ST37 is an interdomain linker of cellobiohydrolase I from the
in which each amino acid was individually substituted by fungus Aspergillus. Its sequence is TSTSKASTTTTSSKTTTT
alanine, in order to study its role on binding activity. Since SSKTTTTTSKTSTTSSSST.6 PT23 is an interdomain linker of
residues 11 and 12 in CEL121 were originally alanine, they endoglucanase A from the bacterium Cellulomonas fimi, and
were replaced instead by glycine. To prepare these mutants has the alternating sequence (PT)11P.7 Both linkers have pre-
and measure their binding activities accurately, GST-peptide viously been applied in recombinant protein and enzyme en-
fusions were constructed by splicing the coding sequences of gineering to separate functional domains. Hence, the protein
CB72, CEL121, and their alanine or glycine-substituted TBP101 consists of CB72::ST37::CEL121, and the protein
mutants into plasmid pGSTf. This resulted in GST-CB72 TBP201 is composed of CB72::PT23::CEL121, as shown in
and GST-CEL121 fusion proteins, as well as 7 GST-CB72 Figure 1A. Within these triblock structures, short 3-amino

Biopolymers (Peptide Science) DOI 10.1002/bip

32 Qi, O’Brien, and Yang

Table II GST-ELISA Binding Assay of Analine-Substituted Affinity Peptides

GST-Peptide OD405a Activity (%) GST-Peptide OD405a Activity (%)

Carbon black binding

GST-CB72 0.566 6 0.022 100 GST-CB72A4 0.554 6 0.081 98
GST-CB72A1 0.444 6 0.052 78 GST-CB72A5 0.383 6 0.036 68
GST-CB72A2 0.459 6 0.024 81 GST-CB72A6 0.534 6 0.033 94
GST-CB72A3 0.594 6 0.030 105 GST-CB72A7 0.493 6 0.046 87
Cellulose binding
GST-CEL121 0.386 6 0.035 100 GST-CEL121A7 0.233 6 0.029 60
GST-CEL121A1 0.357 6 0.034 92 GST-CEL121A8 0.298 6 0.008 77
GST-CEL121A2 0.382 6 0.028 99 GST-CEL121A9 0.278 6 0.018 72
GST-CEL121A3 0.302 6 0.008 78 GST-CEL121A10 0.271 6 0.042 70
GST-CEL121A4 0.321 6 0.040 83 GST-CEL121G11 0.326 6 0.019 84
GST-CEL121A5 0.355 6 0.033 92 GST-CEL121G12 0.315 6 0.013 82
GST-CEL121A6 0.352 6 0.050 91
An average of triplicate assays.

acid hinges consisting of AGG and GGA were placed in the N The 6xHis-TBP101 and 6xHis-TBP201 fusion proteins
and C termini of the linker sequences, respectively, to isolate were produced from pINK101- and pINK201-transformed
the binding peptides from the linkers. An additional peptide BL21 E.coli cells. Since the proteins were not soluble, both
fragment, GSIEGR, was added to the N-termini of these tri- were purified by using a Ni-NTA column under denaturing
block structures to introduce factor Xa recognition site for conditions, and then renatured into solution through dialy-
downstream cleavage of the fusion protein. DNA sequences sis. Freshly purified fusion proteins were subjected to the
coding for such triblock proteins were synthesized and incor- 6xHis-ELISA binding assay to confirm dual binding activity.
porated into pDEST17 and pDEST15. Thus, the resulting One reaction included 0.1 nmol fusion protein and 75 lg
four expression plasmids were pINK101 and pINK201 for carbon black for the pigment binding assay. Sigmacell cellu-
expressing 6xHis-TBP101 and 6xHis-TBP201, and pINK151 lose (150 lg) was used for the cellulose binding assay. A non-
and pINK251 for expressing GST-TBP101 and GST-TBP201, related 42 kDa 6xHis tagged protein YghD was used as a neg-
respectively. Structural schemes for these fusion proteins are ative control. Assay results are shown in Table III. OD405 rep-
shown in Figure 1B. Triblock proteins could be released from resents the binding activity. The results indicate that both
their fusion partner through Xa digestion.8 6xHis-TBP101 and 6xHis-TBP201 have carbon black and cel-
lulose binding activities but the 6xHis-YghD does not. This
demonstrates that TBP101 and TBP201 and not the 6xHis
tags provide dual binding activity. Because the renatured
6xHis-TBP201 aggregated rapidly, its activity was much
lower. Similarly for 6xHis-TBP101, binding activity decreased
quickly because of aggregation. Therefore, in order to

Table III Dual Binding Activity in Selected Triblock Proteins

Carbon Black Binding Cellulose Binding

Fusion Protein Activity (OD405a) Activity (OD405a)

6xHis-ELISA binding assay

6xHis-TBP101 0.350 6 0.065 0.472 6 0.004
FIGURE 1 Recombinant triblock proteins. (A) TBP101 and 6xHis-TBP201 0.054 6 0.002 0.022 6 0.006
TBP201 amino acid sequences are shown in bold letters. The addi- YghD 0.000 6 0.003 0.007 6 0.006
tional GSIEGR fragment is underlined. GenBank accession numbers GST-ELISA binding assay
for their coding sequences were EF562461 and EF562462, respec- GST-TBP201 0.422 6 0.040 0.170 6 0.029
tively. (B) Structural schemes for 6xHis-TBP and GUS-TBP fusion GST 0.018 6 0.020 0.013 6 0.005
proteins. Each element is marked on the top of the scheme. IEGR
indicates factor Xa recognition site. An average of triplicate assays.

Biopolymers (Peptide Science) DOI 10.1002/bip

Recombinant Triblock Protein Dispersant 33

confirm dual binding activity, pINK151 and pINK251 were

transformed into BL21 E.coli cells, and GST-TBP101 and
GST-TBP201 fusion proteins were produced. However, since
GST-TBP101 was insoluble as expressed, purification was dif-
ficult. Only GST-TBP201 could be purified using a glutathi-
one-agarose column and subjected to a GST-ELISA binding
assay that included 0.1 nmol fusion protein and 75 lg carbon
black to assay for pigment binding or 150 lg Sigmacell cellu-
lose to assay for the cellulose binding. Free GST was used as
control. ELISA results (Table III) confirmed that TBP201
exhibited strong dual binding activity even though it was
fused with a much larger GST protein sequence.

TBP201 Disperses Carbon Black Pigment

and Binds It to Paper
Although the dual binding activity of peptides TBP101 and
TBP201 was confirmed, this did not prove that they would FIGURE 2 TBP201 disperses carbon black pigment. (A) In a
standard dispersion reaction, carbon black pigment was mixed with
function as pigment dispersants or binders. To demonstrate TBP201, dotted on a glass slide, and observed for flocculation. In
these functionalities, GST-TBP201 was treated with factor Xa the controls, TBP201 was substituted by BSA, GST, and protein-free
to release free TBP201. TBP201 was then purified as buffer, respectively. (B) Dispersion reaction was modified to include
described earlier and used for further functional studies. TBP201 protein as dispersant and CB72 peptide as competitor.
Sand milling and accelerated laundering are common Reactions containing either TBP201 or CB72 were used as controls,
respectively. (C) Dispersion reactions were assembled as described
techniques to study dispersion and binding properties,
in (B), except that GST was used as dispersant. In every panel, pro-
respectively, for traditional chemical-based dispersants and teins and peptides used in each assay reaction were marked on the
binders.9,10 However, because these methods require a large top of panel.
amount of protein, microscale dispersion and attachment
assays were developed and used in these studies. To evaluate GST is substituted for TBP201 in the dispersion reaction, it
the dispersant properties of TBP201, 10-lL mixtures con- functions as a carbon black dispersant as well. Since GST
taining 50 lg carbon black and various amounts of TBP201 does not possess specific carbon black binding activity
(0–0.6 nmol) were prepared. After gentle mixing, 5 lL of (Table III), its mechanism of dispersion is probably different
each suspension was dropped onto a glass slide, allowed to than that of TBP201. To test this hypothesis, a competition
stand for 2 min, and then observed for pigment dispersion. experiment with carbon black was conducted. Compared to
The results indicated that increasing the amount of TBP201 the regular dispersion assay described earlier, the competition
improved pigment dispersion. A complete dispersion was experiment contained no glycerol and used much less protein
observed when TBP201 reached 0.1 nmol and beyond (data in order to render the pigment dispersion easily reversible.
not shown). Based on this observation, a standard dispersion Specifically, just 0.1 nmol TBP201 protein was mixed with
assay with well-designed controls was conducted. A 10-lL 50 lg carbon black in a 10-lL reaction. As observed in the
reaction contained 0.6 nmol TBP201 to ensure complete dis- experiment described earlier, the smaller amount of TBP201
persion, or contained 0.6 nmol BSA, GST, and no protein as was nonetheless sufficient to disperse carbon black (Figure 2B).
controls, respectively. As shown in Figure 2A, the carbon When 0.7 nmol synthetic CB72 peptide was included as a
black pigment was finely dispersed when TBP201 was added. competing peptide, it blocked the ability of TBP201 to
However, samples with 0.6 nmol BSA or with no protein disperse pigment, resulting in flocculation. Similarly floccu-
showed flocculation of the carbon black (Figure 2A). These lation also occurred in a negative control that had 0.7 nmol
results suggest that TBP201 functions as a carbon black dis- synthetic CB72 but lacked TBP201 (Figure 2B). The results
persant in water. Since attaining a dispersion did not require of these competition experiments show that the carbon black
milling or grinding, the interaction between TBP201 and car- binding activity of the CB72 peptide is directly involved in
bon black is apparently strong enough to mediate particle TBP201’s activity as a pigment dispersant. When a similar
self-dispersion. This is a property that incumbent dispersants competition test was conducted, using 0.1 nmol GST instead
typically do not offer. As shown in Figure 2A, if 0.6 nmol of of TBP201, CB72 failed to block the dispersant function of

Biopolymers (Peptide Science) DOI 10.1002/bip

34 Qi, O’Brien, and Yang

GST (Figure 2C). This suggests that pigment dispersion

by GST does not depend on the specific activity of CB72, and
is thus operating by a different mechanism. Because of a lack
of glycerol in the competition tests, flocculation of carbon
black is much more severe in Figures 2B and 2C than that in
Figure 2A.
To determine whether or not TBP201 could attach carbon
black to a cellulose surface, 50 lg carbon black was added to
a 15-lL attachment assay containing 0.6 nmol TBP201 pro-
tein. In two negative control experiments, 0.6 nmol GST and
BSA were used to replace TBP201, respectively. After a 20-
min incubation, 5 lL of the mixture was spotted on a piece
of Whatman 5 filter paper. The paper absorbed the water
quickly and left carbon black on the surface to allow direct
interaction between the paper, pigment, and triblock pro-
tein. The paper was then washed in 10 mL TTBS solution,
and the pigment attachment was evaluated. The results
(Figure 3A) show that carbon black attaches to the paper
surface when the formulation includes TBP201, but attach-
FIGURE 3 TBP201 enhances carbon black pigment attachment
ment does not occur when TBP201 is replaced with GST or
to paper surface. (A) In a standard attachment assay, carbon black
BSA. Similar results were obtained from the assay using pigment was mixed with TBP201, dotted on filter paper, and then
100% cotton fabric as the cellulosic surface (data not washed. Pigment retained on the paper before and after washing
shown). As anticipated, the CB72 and CEL121 elements was recorded. In the controls, GST and BSA were used to replace
interact independently with carbon black and cellulosic sur- TBP201, respectively. (B) In an attachment assay for the CB72 com-
petition test, pigment was added with CB72 peptide, prior to mix-
face, respectively. Both carbon black and cellulose binding
ing with TBP201. A standard attachment assay was used as control.
activity should contribute to its function as a binder. To (C) In an attachment assay for the CEL121 competition test,
demonstrate this, competition tests for paper attachment CEL121 peptide was mixed with the pigment, together with
were conducted. In a test using CB72 as a carbon black TBP201. A standard attachment assay was used as control. In every
binding competitor, 50 lg carbon black and 8.2 nmol syn- panel, proteins and peptides used in each assay reaction were
thetic CB72 peptide were mixed in 10 lL IEB buffer for marked on the top of panel. BW indicates the records taken before
washing. AW indicates the records taken after washing.
10 min. The resultant formulation was then added to 5 lL IEB
solution containing 0.6 nmol TBP201 protein. After 20 min,
5 lL of the mixture was spotted on a piece of Whatman 5 fil- through aqueous interactions in the ink formulation. In
ter paper. The paper was washed in TTBS. Carbon black TBP201, the PT23 linker isolated CB72 and CEL121 and sup-
attachment before and after washing was evaluated. In the ported dual binding activity. Yet there is no evidence to sug-
test using CEL121 as the print surface binding competitor, gest that the linker itself is also directly involved in pigment
carbon black was simultaneously mixed with 8.2 nmol syn- dispersion and/or binding. To investigate this, the PT23 cod-
thetic CEL121 peptide and 0.6 nmol TBP201 protein in ing sequence in pINK251 was replaced with shorter ones,
15 lL IEB buffer. The results of both competition experiments resulting in pINK2a51, pINK2b51, and pINK2c51. These
are shown in Figures 3B and 3C. The tests without competi- plasmids coded for GST-TBP2a01, GST-TBP2b01, and GST-
tors served as the positive controls. These results show that TBP2c01 fusion proteins. They were similar to GST-TBP201,
free CB72 and CEL121 peptides blocked, at least partially, but had PT11, PT5, and PT3 linkers. Sequences for these
pigment attachment. Hence, dual binding activity was con- linkers were (PT)5P, (PT)2P, and PTP, respectively, as sum-
firmed by the ability of TBP201 to bind to print media. marized in Table IV.
GST-TBP201 and its derivatives were expressed in BL21
E. coli cells and purified by using a glutathione-agarose col-
Linker Size Affects TBP201’s Functions umn, as described earlier. Dual binding activities were tested
By design, a linker sequence in a TBP dispersant not only using the GST-ELISA binding assay. One assay included 0.1
functions as an isolating element between binding peptides, nmol GST-TBP fusion protein and 37.5 lg carbon black for
but also plays a key role in stabilization of the dispersion measuring carbon black binding activity or 600 lg Sigmacell

Biopolymers (Peptide Science) DOI 10.1002/bip

Recombinant Triblock Protein Dispersant 35

Table IV Dual Binding Activity of GST-TBP201 and Selected Derivatives

Carbon Black Binding Cellulose Binding

Fusion Protein Linker OD405a Activity (%) OD405a Activity (%)

GST-TBP201 (PT)11P 0.522 6 0.023 100 0.125 6 0.027 100

GST-TBP2a01 (PT)5P 0.632 6 0.085 121 0.125 6 0.028 100
GST-TBP2b01 (PT)2P 0.736 6 0.025 141 0.179 6 0.059 143
GST-TBP2c01 PTP 0.946 6 0.043 181 0.219 6 0.027 175
An average of triplicate GST-ELISA assays.

cellulose for measuring cellulose binding activity. Assay interact with substrates. It has been applied to identify bind-
results are shown in Table IV, in which the activity is pre- ing peptides for a wide range of molecules, such as proteins,
sented as OD405. Results indicated that all evaluated PT link- nucleic acids, and small molecules.11–13 This study and those
ers supported dual binding activity. However, the shorter of several other groups have shown its utility for selecting
linkers promoted higher binding activity for both carbon peptides that recognize and bind to solid surfaces and par-
black and cellulose. To test the effects of linker size on dis- ticles.14–16 Some of these peptides have been incorporated
persant and surface binding functions, GST-TBP201 and into new biomaterials that offer novel interfacial binding
related short versions were cleaved by factor Xa to release functionalities.16–18 Because the substrates are solids, it is a
TBP201, TBP2a01, TBP2b01, and TBP2c01. The resultant tri- challenge to characterize peptide candidates for their binding
block proteins were purified and used in the dispersion and properties. To address this, we developed a GSTf fusion and
attachment assays described earlier. The pigment dispersion assay system to link affinity peptides to GST protein, inde-
results (Figure 4A) indicate that TBPs with PT23, PT11, and pendent of the phage, and to precisely measure and compare
PT5 linkers dispersed pigment equally well. TBP2c01, which their substrate-specific binding activities. By combining this
had a PT3 linker, did not disperse carbon black but rather system with alanine-scanning, we were able to confirm the
caused flocculation to occur. For the attachment assay, car- binding activities of carbon black and cellulose binding pep-
bon black attachment on Whatman 5 filter paper before and tides. In addition, the contribution of each residue to binding
after washing is shown in Figure 4B. Specifically, TBP201, activity was determined for both sequences. Our results sug-
which had a PT23 linker, gave the strongest attachment. gest that the three critical residues (F1, H2, and W5) contrib-
Attachment became weaker and the pigment was easier to uting to the carbon black binding activity of CB72 are aro-
wash off when the size of PT linker was reduced. Hence, the
linker strongly impacted dual binding activity and dispers-
ant/binder functionality. Within the size range tested in this
study, the shortened PT linkers enhanced dual binding activ-
ity but reduced the efficiency of pigment dispersion and
attachment. Although it provided a dual binding activity, the
PT3 linker in TBP2c01, the smallest one tested here, per-
formed poorly in the dispersion and attachment assays.
These data suggest that, besides functioning as an isolating
spacer, the PT linker is also directly involved in pigment dis-
persion and attachment. While the size of the PT linker does
FIGURE 4 Pigment dispersion and printing medium binding
not restrict dual binding activity, the longer linker enhances
properties of TBP201’s derivatives. (A) Standard dispersion assay
pigment dispersion and paper binding properties in the tri- was conducted as described in Figure 2. TBP201 and its short linker
block protein. Thus, PT23 is the linker of choice among derivatives were used in assay reactions, respectively, as marked on
those evaluated in this study. the top of panel. A reaction containing no protein was set up as a
control. (B) Standard attachment assay was conducted as described
in Figure 3. TBP201 and its short linker derivatives were used in
assay reactions, respectively, as marked on the top of panel. A reac-
DISCUSSION tion containing no protein was set up as control. BW indicates the
Biopanning is a powerful high throughput technique to iden- records taken before washing. AW indicates the records taken after
tify peptides from phage display libraries that specifically washing.

Biopolymers (Peptide Science) DOI 10.1002/bip

36 Qi, O’Brien, and Yang

matic amino acids, while three out of five critical residues sible to shorten the binding domains, a smaller linker
(K3, Q7, and R8) contributing to cellulose binding activity of sequence is important to keep triblock protein compact for
CEL121 have polar side chains. These findings suggest that greatest efficiency. Second, it has high degree of conforma-
hydrophobic interactions are involved in carbon black bind- tional rigidity by nature of alternating proline residues. A
ing to CB72. In contrast, cellulose-binding activity in rigid linker can serve as an insulator between the binding
CEL121 is probably controlled by ionic interactions. How- domains and, therefore, support dual binding activity.
ever, it would not be surprising if other peptides bind to their Finally, PT23 is very hydrophilic because of its proline and
substrates through mechanisms different from CB72 or threonine content, allowing it to interact directly with water
CEL121. Because of the complexity of the solid surfaces in to form stable pigment dispersions. In these experiments,
those studies, the mode of binder receptor interaction could PT23 was demonstrated to function in the triblock protein as
be very different. Hence, enhanced binding functions of pro- designed. The fact that the dual binding activity favors a
tein-based dispersants may be achieved by incorporating shorter PT linker while pigment dispersion/attachment func-
multiple binding peptides into a single triblock protein mol- tions favor longer ones suggests that the linker is involved in
ecule or by combining several triblock proteins containing both domain isolation and pigment dispersion/attachment
different binding peptides. through different mechanisms.
In this work, the triblock protein TBP201 contained
CB72::PT23::CEL121. Since the CB72 and CEL121 peptide
We thank Dr. Mark Nelson for support and encouragement; Dr.
sequences are isolated from each other by a linker, the mole- Harry Spinelli, Dr. David Deters, Dr. Ron Hoess, and Dr. Hong
cule exhibits carbon black and cellulose binding activities. Wang for helpful discussions; Cathy Byrne, Mike Madden, and Ray
Dual binding activity is essential for TBP201 to disperse pig- Jackson for assistance with biopanning and DNA sequencing.
ment and attach pigment to a printing surface. Free CB72
and CEL121 can destroy these functions by competing with
TBP201 for the binding sites. This sequence-dependent func-
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Biopolymers (Peptide Science) DOI 10.1002/bip