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Journal of Biotechnology
Abstract Volume 160, Issues 3–4, 31 August 2012, Pages 222-228 Structural and Functional Analysis
of the Signal-Transducing Linker …
Keywords
the pH-Responsive
Journal One-Volume 427, I…
of Molecular Biology,
1. Introduction
Screening of PC and PMMA-binding Component
Sophie Buchner,System CadC
…, Kirsten Jung of

2. Materials and methods Escherichia

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3. Results and discussion peptides for site-specific Applications of LEDs in optical

4. Conclusion immobilization of proteins sensors and chemical sensing…
device forand
Renewable detection of Energy Review…
Sustainable
Acknowledgement
Yoichi Kumada a , Sho Murata b, Yasuyuki Ishikawa a, biochemicals,
Pulin heavy
Yeh, …, Ting-Jou Dingmetals, and
References Kazuki Nakatsuka b, Michimasa Kishimoto a environmental
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A handheld laser-induced
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Cited by (21) applications
Talanta, Volume 150, 2016, pp. 135-141
https://doi.org/10.1016/j.jbiotec.2012.02.010 Get rights and content
Xiao-Xia Fang, …, Qun Fang

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Abstract
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In the present study, we used proteomic research technology to
develop a method for the screening and evaluation of material-binding
peptides for protein immobilization. Using this screening method,
soluble Escherichia coli proteins that preferentially adsorbed onto
polycarbonate (PC) and poly(methylmethacrylate) (PMMA) as model
plastic materials were first isolated and identified by 2-dimensional
electrophoresis (2DE) combined with peptide mass fingerprinting
(PMF). The genes of identified protein candidates (ELN, MLT, OMP, and
BIF) that exhibited a hexahistidine tag (6×His-tag) were over-expressed
by E. coli BL21 (DE3), and the proteins were purified by IMAC affinity
chromatography. The candidates for PC and PMMA-binding peptides
were isolated from peptide fragments from affinity protein candidates,
which were digested with trypsin and chymotrypsin. Consequently, 5
candidates for the PC-binding peptide and 2 candidates for the PMMA-
binding peptide were successfully identified by MALDI-TOF MS. All of
the peptides identified were introduced to the C-terminus of
glutathione S-transferase (GST) as a model protein for immobilization.
Adsorption of peptide-fused and wild-type GSTs onto the plastic
surfaces was directly monitored using a quartz crystal microbalance
(QCM) device. Consequently, genetic fusion of PC-MLT8 and PC-OMP6
as PC-binders and PM-OMP25 as a PMMA-binder significantly
enhanced the adsorption rates of GST, achieving an adsorption density
that was more than 10 times higher than that of wild-type GST.
Furthermore, the residual activity levels of GST-PC-OMP6 and GST-PM-
OMP25 in the adsorption state were 2 times higher than that of wild-
type GST. Thus, the PC and PMMA-binding peptides identified in this
study, namely PC-OMP6 and PM-OMP25, were considerably useful for
site-specific immobilization of proteins, while maintaining a higher
adsorption density and residual activity levels. The method
demonstrated in this study will be applicable to the isolation of a
variety of material-binding peptides against the surfaces of unique
materials.

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Keywords
Affinity peptide tag; Protein immobilization; Proteome analysis

1. Introduction
Recently, immobilization technologies of functional proteins onto the
surfaces of organic and inorganic solid materials have been recognized
to be important for the construction of protein-based nano-reactors,
micro-fluidics, and microarray chips. Although a variety of
immobilization methods including physical adsorption, chemical
coupling, encapsulation, and others have been proposed, there is still
much room for improvement to achieve higher density and biological
activity of proteins immobilized to the greatest possible extent (Zhang
et al., 2005, Butterfield et al., 2001, Lutz et al., 1990, Nakanishi et al.,
2008, Rusmini et al., 2007, Hwang et al., 2007).

Universal affinity peptides for purification, detection, and

immobilization such as 6×His tag, FLAG-tag, c-myc-tag, and Strep-tag,
are now essential for biochemical research in laboratories (Wang et al.,
2001, Carlsson et al., 2000, Hedhammar et al., 2005). Their
performance strongly depends on the density and stability of ligand
molecules such as Ni-NTA, monoclonal antibody, and streptavidin that
were introduced on the surfaces of solid supports. In particular, when
the ligand molecules are proteins, not only their density but also their
residual activity in an immobilized state directly affect the density of
target proteins that are genetically fused with affinity peptide tags.

However, several peptides that directly recognize the surfaces of solid

materials have been isolated, and they are expected to be useful as
alternative affinity peptide tags for direct immobilization of target
proteins onto solid support surfaces (Sarikaya et al., 2003, Shiba, 2010).
This research group has also developed and characterized affinity
peptide tags that directly recognize the surface of hydrophilic
polystyrene (PS-tag). Genetic fusion of the PS-tag to several target
proteins such as glutathione S-transferase (GST), o-acetylserine
sulhydrylase A (OASS A), and single-chain Fv antibodies (scFv) resulted
in site-specific immobilization of proteins with a much higher density
and residual activity (Kumada et al., 2006, Kumada et al., 2007, Kumada
et al., 2009a, Kumada et al., 2009b, Kumada et al., 2010a, Kumada et al.,
2010b).

Most material-binding peptides reported so far were isolated from

random peptide libraries in which genetically engineered phage
particles or Escherichia coli cells displayed oligo peptides on their
surfaces. Although biopanning using such peptide libraries is becoming
a standard procedure for identification of peptides that bind to target
substances such as antibodies, enzymes, receptors, and others, this
method often makes it difficult to isolate material-binding peptides,
mainly due to the following 3 reasons: (1) non-specific and multipoint
interaction of phage particle or cells, and/or different growth rates of
each clone prohibit efficient fine screening; (2) the material-binding
peptides often have lower sequence-specific motifs compared to
protein-binding affinity peptides; and, (3) there are no guidelines as to
how many rounds of biopanning should be performed to isolate
material-binding peptides. Therefore, even when complicated and
laborious biopanning procedures are repeated many times, the amino
acid sequences of the peptides finally obtained often lack pattern and
tendency. When polystyrene-binding peptides (PS-tag) were isolated
from a random peptide display E. coli cell library by this research group,
the biopanning procedure had to be repeated 10 times. Consequently,
there was no typical motif or tendency with the amino acid sequences
of the isolated peptides. All the peptides selected were then genetically
fused with glutathione S-transferase (GST), and the adsorption
properties of peptide-GST fusion proteins had to be characterized in
order to determine which peptide possessed the desired binding
affinity for the surface of polystyrene.

Because of the situation described above, we developed and

demonstrated a new screening method to identify the amino acid
sequences of material-binding peptides by a combination of proteome
analysis technologies such as 2-dimensional electrophoresis (2DE),
MALDI-TOF MS and HPLC. Fig. 1 schematically shows the
comprehensive screening procedure in our method. E. coli cells contain
more than 4000 kinds of proteins that possess different and unique
amino acid sequences. Some of them may preferentially adsorb onto
the surfaces of certain materials with a relatively strong binding
affinity. In our screening method, proteins that are preferentially
adsorbed onto the target materials with a strong binding affinity were
first isolated from the soluble fraction of E. coli cell lysate and identified
by 2DE followed by peptide mass fingerprinting using MALDI-TOF MS.
The proteins that over-expressed and purified were digested with
trypsin or chymotrypsin, and the peptide fragments that maintained a
high binding affinity were identified by HPLC followed by MALDI-TOF
MS. Finally, the adsorption and residual activity levels of peptide-fused
proteins were compared in order to evaluate the usefulness of the
identified peptides as affinity peptide tags for protein immobilization.
E. coli internal proteins as a protein source were used in this study due
to the following reasons. (1) E. coli proteins with few post-translational
processing such as glycosylation can be easily identified by peptide
mass fingerprinting method. (2) Genome database as well as protein
database is well-studied and established. Therefore, the genes
identified from 2D-gel can be easily cloned into the expression vector.
(3) The genes of E. coli proteins can be easily expressed in the original
host cell without codon optimization.

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Fig. 1. Screening procedure of material-binding peptides developed in

this study.

In this study, polycarbonate (PC) and poly(methyl methacrylate)

(PMMA) were used as model solid materials, and the PC-binding and
PMMA-binding peptides were screened by the method described
above.

2. Materials and methods

2.1. Materials
Cube-shaped plastic grains made of polycarbonate (PC) and poly
(methylmethacrylate) (PMMA) were purchased from Teijin (#AD-5503)
and Asahi-Kasei Chemicals (#70NH), respectively. Packed columns of
affinity chromatography resins, HisTrap HP, and GSTrap HP were
purchased from GE Healthcare. The octadecylsilane (ODS) column,
5C18-AR-II for HPLC analysis was from Nacalai Tesque. The expression
vector pET 22b from Merk was used for expression of cloned affinity
protein candidates. Other chemicals and materials obtained from
Nacalai Tesque, Wako Pure Chemicals, and Sigma–Aldrich were of
reagent grade unless otherwise specified.

2.2. Extraction of soluble protein from E. coli

E. coli BL21 (DE3) was used as a model host strain in this study. A single
colony was inoculated into 20 ml of LB medium in a 100-ml flask, and
the flask was shaken at 37 °C overnight. Then, a portion of the culture
was added to 100 ml of 2×YT medium in a 500 ml shake flask to obtain
a final OD600 of 0.1. The flask was shaken at 200 rpm and 37 °C for 7 h.
The cells were harvested by centrifugation at 10,000 × g, and the
supernatant was removed. Pellets were suspended with 10 ml of
Bugbuster™ protein extraction reagent (Merk) containing 5 µl of
benzonase nuclease (Merk) and 100 µl of protease inhibitor cocktail
(Nacalai), and the mixture was incubated at 37 °C for 30 min. After
centrifugation for 20 min at 10,000 × g, the supernatant was recovered
as an E. coli protein sample and stored at −20 °C until use.

2.3. Screening and identification of proteins

preferentially adsorbed onto the surface of plastic grains
E. coli protein samples in 5 ml portions were mixed with 10 g of plastic
grains with the surface areas of 200 cm2 (PC) and 183 cm2 (PMMA) in
glass vials. The glass vials were shaken at 80 rpm and 25 °C for 5 h. The
plastic grains were washed 3 times by 40 ml of PBS for 5 min followed
by washing 3 times with 40 ml of milli-Q water. The plastic grains were
transferred to other glass vials, and then proteins adsorbed on the
plastic surfaces were eluted by addition of 4 ml of solubilization buffer
containing 6 M urea, 4 % (w/v) CHAPS, 2% (v/v) pharmalyte pH 3–10 (GE
Healthcare), and 10 mg/ml DTT. The vials were shaken at 160 rpm and
25 °C for 30 min, and then the supernatants were recovered as an
affinity protein sample. The sample was concentrated 5-fold by
ultrafiltration using MICROCON YM-10 (millipore). The affinity protein
samples finally obtained were directly applied to isoelectric focusing
(IEF) gel followed by SDS-PAGE 2-dimensional electrophoresis (2DE).
The 2DE gels were stained with coomassie brilliant blue (CBB), and the
gels of visualized protein spots were collected. The gels were incubated
for 45 min in 150 µl of 0.1 M ammonium bicarbonate washing solution
containing 40% (v/v) acetonitrile followed by washing with 500 µl of
milli-Q water. This washing step was repeated, and then the
supernatant was removed. The gels were mixed with 200 µl of the
washing solution and incubated for 5 min. After removal of the
supernatant, the gels were incubated in 100 µl of acetonitrile for 5 min.
The supernatant was removed and the gels were then dried in a freeze
dryer. The gels were swollen by the addition of 5 µl of modified trypsin
solution (Promega), placed on ice for 20 min, and then incubated
overnight at 37 °C. The swollen gels were mixed with 50 µl of an
extraction solution containing 50% (v/v) acetonitrile and 1% (v/v) TFA
followed by gentle stirring for 30 min. After centrifugation, the
supernatant was collected. The same extraction procedure was
repeated using 25 µl of the extraction solution. The supernatant
collected by 2 extraction procedures was mixed and then lyophilized.
The lyophilized peptides were dissolved by adding 2 µl of the
extraction solution. The solution of dissolved peptides was mixed with
saturated α-cyano-4-hydroxy-cinnamic acid (CHCA) solution
containing 70% (v/v) acetonitrile and 0.1% (v/v) TFA, and 1 µl of the
mixture was applied to a target plate (BRUKER Daltonics). The plate
was incubated at room temperature for more than 15 min. The mass
spectrum of peptides was detected by an Autoflex Speed TOF/TOF mass
spectrometer (BRUKER Daltonics). The names of affinity protein
candidates were determined by the mascot search engine (MATRIX
SCIENCE, http://www.matrixscience.com/ ) utilizing the mass
spectrum data obtained.

2.4. Cloning and expression of affinity protein genes

Information on the DNA sequences of the encoding affinity protein
candidates that were identified by the PMF method was obtained from
the genome database of E. coli BL21 on the NCBI website
(http://www.ncbi.nlm.nih.gov/ ). Genomic DNA of the E. coli strain was
extracted using a Wizard genomic DNA purification kit (Promega). The
genes of the affinity proteins with the 5′ and 3′ restriction sites of Nde I
and Not I, respectively, were amplified by PCR using genomic DNA as a
template. The genes were cloned into the expression vector pET22b (+)
between the recognition sites of the restriction enzymes, Nde I and Not
I. Consequently, the genes of the affinity proteins were inserted
between the start and stop codons in the pET22 vector, and the target
proteins could be produced as fusion proteins with a hexahistidine-tag
(6×His-tag) at their C-termini. The host strain of E. coli BL21 (DE3) was
transformed with these expression vectors, and the transformants were
then selected on the LB agar plate containing Amp. The selected cells
were precultured overnight in 10 ml of LB medium containing Amp. A
portion of the culture was added to 50 ml of Overnight Express TB
medium supplemented with Amp and Cam in a 500 ml shake flask to
obtain a final OD600 of 0.1. The flask was shaken at 200 rpm and 30 °C
for 24 h. According to SDS-PAGE analyses of the soluble and insoluble
cellular proteins, all of the protein candidates were located mainly in
insoluble fractions. Therefore, the inclusion bodies containing the
affinity protein candidates were purified by the method reported
previously (Kumada et al., 2010b). Briefly, cell pellets were disrupted by
the addition of 5 ml of Bugbuster™ containing 1 mg/ml lysozyme and
0.05% (v/v) benzonase nuclease (Merk) followed by centrifugation at
10,000 × g for 15 min. Insoluble aggregates were collected by
centrifugation for 15 min at 10,000 × g and 4 °C and washed twice with
distilled water. They were then dissolved in a denaturation buffer (pH
7.2) containing 6 M guanidine–HCl, 10 mM mercaptoethanol, 300 mM
NaCl, and 40 mM sodium phosphate. After removal of cell debris by
centrifugation, the supernatant was directly applied to 5 cm3 of
HisTrap HP affinity column (GE HealthCare) equilibrated with buffer A
(pH 7.2) containing 8 M urea, 300 mM NaCl, 40 mM sodium phosphate,
and 40 mM imidazole. After washing the column with buffer A, the
bound proteins were eluted with buffer B (pH 7.2) containing 8 M urea,
300 mM NaCl, 40 mM sodium phosphate, and 400 mM imidazole. The
eluate was dialyzed against PBS containing 8 M urea. Protein
concentration in the eluate was determined by DC-protein assay (Bio-
Rad Laboratories) using BSA dissolved in 8 M urea-PBS as a standard.

2.5. Identification of affinity peptide fragments from

affinity proteins
Affinity proteins in 8 M urea-PBS were diluted with PBS to a protein
concentration of 0.5 mg/ml and at a urea concentration of less than
1 M. Then 20 µg of trypsin or chymotrypsin (Promega) was dissolved
with the protein solutions, and the mixture was incubated at 37 °C for
12 h to produce peptide fragments derived from affinity proteins. Next,
600 µl portions of the peptide samples were mixed with 16 g of plastic
grains (PC 320 cm2, PMMA 293 cm2), and then the mixture was shaken
at 200 rpm and 25 °C for 2 h. Peptide components in the solution
before and after adsorption were analyzed by HPLC using a 5C18-AR-II
packed column (i.d. 4.6 mm × 150 mm). The peptides with peak areas
that were decreased by more than 70% after adsorption were
fractionated, followed by lyophilization. The molecular weight of the
isolated peptides was determined by MALDI-TOF MS or MS/MS
analysis, and then amino acid sequences of the peptide fragments were
determined by matching them with the original amino acid sequences
of affinity proteins.

2.6. Preparation of glutathione S-transferase genetically

fused with peptide tags
All identified candidates for affinity peptide tags were genetically fused
with the model enzyme, glutathione S-transferase (GST) at the C-
terminus by the method reported previously. Briefly, oligo nucleotides
encoding DNA sequences of peptides and restriction sites were
chemically synthesized. Sense and antisense oligo DNAs in 100-µl
portions were mixed in H buffer to a final concentration of 10 µM. The
mixture was incubated at 95 °C for 5 min, and then the temperature
was gradually decreased from 95 °C to room temperature (∼25 °C) in
order to anneal them correctly. The double-strand DNAs prepared were
collected by ethanol precipitation and then digested with restriction
enzymes Bam HI and Eco RI. The fragments were purified by ethanol
precipitation and ligated into the GST expression vector pGEX-3X
between the restriction enzymes sites of Bam HI and Eco RI. E. coli BL21
(DE3) cells were transformed with the expression vectors constructed,
and then the transformants were inoculated into 10 ml of 2×YT
containing Amp in a 200-ml shake flask. The flask was shaken
vigorously overnight at 37 °C. The culture was inoculated into 50 ml of
2×YT containing Amp in a 500-ml shake flask to obtain a final
OD600 = 0.1. The flask was shaken at 200 rpm and 37 °C until the value
of OD600 reached approximately 1.0. Next, 5 µl of 1 M IPTG was added,
and the flask was shaken at 25 °C and 200 rpm for 18 h. The cells were
harvested by centrifugation and lysed by the addition of 5 ml of
Bugbuster™ containing 1 mg/ml lysozyme and benzonase nuclease.
The cellular soluble fraction was applied to a 5 ml GSTrap HP column
(GE Healthcare) pre-equilibrated with PBS containing 1 mM DTT, and
bound peptide-fused GSTs were eluted by gradient elution with 50 mM
Tris–HCl containing 100 mM of reduced glutathione. The eluates
containing peptide-fused GSTs were dialyzed against PBS followed by
quantification of protein concentration by DC protein assay using BSA
as a standard protein.

2.7. Adsorption monitoring of peptide-fused GSTs onto

polymers by quartz crystal microbalance
A quartz crystal microbalance (QCM) device (Affinix QN) operating at
27 MHz was used for monitoring the adsorption process of peptide-
fused GSTs. PC and PMMA grains were dissolved in chloroform to a
concentration of 1% (w/v). A single droplet of the polymer-dissolved
solvent was put on the surface of the sensor chip, followed by spin-
coating at 6000 rpm for 1 min to prepare the polymer thin film on the
gold surface of the sensor chip. The polymer-coated sensor chip was
placed on the QCM device and incubated in 8 ml PBS at 25 °C with
gentle stirring at 1000 rpm until the frequency signal became almost
constant. The GST solutions were then added to obtain a final GST
concentration of 1 µg/ml, and frequency signals were monitored for
60 min.

2.8. Activity measurement of peptide-fused GSTs in the

adsorption state
Specific activities (Spsol) of wild-type GST and peptide-fused GSTs in
solution were determined according to the method reported previously
(Kumada et al., 2006). Activity of GSTs in the adsorption state was
determined by the following method. PC or PMMA grains with a total
surface area of 28 cm2 were soaked in 3 ml of 100 µg/ml GST solution
and incubated at 25 °C for 2 h with gentle stirring. Then, the
supernatant was corrected, and the concentration of GST in the
supernatant was determined by DC protein assay (Bio-Rad
Laboratories) to determine the amount (Qad, mg) of GST that was
immobilized. The surfaces of plastic grains were gently washed 3 times
with PBS, and then mixed with 3 ml of a substrate solution containing
10 mM GSH, 10 mM CDNB, and 0.1 M potassium phosphate buffer (pH
6.5). The solution was gently stirred at 25 °C, and change in absorbance
at 340 nm was measured with a NanoDrop spectrophotometer (Thermo
Fisher Scientific) at 30-s intervals. Activity (Aad, U) of GST in the
adsorption state was determined from the slope of the absorbance
change. The specific activities (Spad, U/mg) of GSTs in the adsorption
state, and residual activity (R, %) were determined using the following
equations.

3. Results and discussion

3.1. Isolation and identification of affinity protein

candidates from soluble E. coli protein samples
We first isolated and identified E. coli internal proteins that physically
adsorbed with strong binding affinity onto the PC and PMMA grain
surfaces by 2-dimensional electrophoresis (2DE), followed by peptide
mass fingerprinting (PMF). Fig. 2(a)–(c) shows results of 2DE images of
E. coli internal proteins before adsorption (Fig. 2(a)), and those eluted
from the PC and PMMA grains (Fig. 2(b) and (c)), respectively.
Interestingly, the position of protein spots eluted from the PC and
PMMA grains were very similar. As shown in Table 1, 4 kinds of E. coli
proteins, namely, elongation factor Tu (ELN), Omp F porin (OMP), malto
porin (MLT), and bifunctional aconitate hydratase (BIF) were identified
as affinity protein candidates, indicating that those 4 proteins
preferentially adsorbed onto the surfaces of both PC and PMMA even in
the presence of other E. coli internal proteins as competitors. These
results imply that these proteins may have amino acid sequences with
a high binding affinity for PC and/or PMMA.

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Fig. 2. Two-dimensional electrophoresis of E. coli proteins. (a) Lysate of

E. coli cells, (b) proteins isolated from PC surface, (c) proteins isolated
from PMMA. Protein's number were extracted from the gels and
identified by the PMF method as shown in Table 1.

Table 1. Affinity protein candidates identified from E. coli cell lysate.

No. Name of protein pI M.W.

1 OMP F porin (OMP) 4.69 39.3 kDa

2 Malto porin (MLT) 4.85 50.0 kDa

3 Bifunctional aconitate hydratase (BIF) 5.32 95.1 kDa

4 Elongation factor Tu (ELN) 5.30 43.3 kDa

3.2. Cloning and production of affinity protein candidates

from E. coli genomic DNA
There were 4 identified affinity protein candidates which were cloned
into the commercially available expression vector, pET22b (+) between
the sites of restriction enzymes, Nde I and Not I. Those proteins that
were fused with a 6×His-tag at their C-termini were produced by
cultivation of the transformants using the Overnight Express
Autoinduction System (OE system). Although all of the protein
candidates produced were expressed mainly in insoluble fractions as
inclusion bodies, they were successfully solubilized with 6 M
guanidine–HCl and then purified by affinity chromatography using a
His-Trap HP column in the presence of 8 M urea, as shown in Fig. 3.

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Fig. 3. SDS-PAGE of affinity protein candidates after purification. Lane 1:

OMP F porin (OMP), Lane 2: malto porin (MLT), Lane 3: bifunctional
aconitate hydratase (BIF), Lane 4: elongation factor Tu (ELN).

After the refolding of these protein candidates using the step-wise

dialysis method (Umetsu et al., 2003), the proteins were digested with
trypsin or chymotrypsin to produce peptide fragments.

3.3. Isolation and identification of peptide fragments

showing binding affinity toward plastics
The peptides produced from the affinity protein candidates by
digestion with trypsin or chymotrypsin were adsorbed onto the
surfaces of the plastic grains. Fig. 4 shows the typical results of HPLC
analysis using peptide fragments derived from OMP before and after
adsorption onto the surfaces of the PC grains. These results indicate
that peaks of peptides with the ability to bind to the plastic surface
drastically decreased after adsorption. In this study, a peptide with a
peak area that was decreased by more than 70% after adsorption was
defined as an affinity peptide candidate.

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Fig. 4. HPLC analysis of peptide fragments derived from OMP before and
after adsorption. (a) Peptides digested with trypsin, (b) peptides
digested with chymotrypsin. Peptides derived from affinity protein
candidates with trypsin or chymotrypsin were adsorbed onto the
surfaces of plastic grains (PC 320 cm2, PMMA 293 cm2). Then the
concentration change in each peptide component before and after
adsorption was compared by HPLC using a 5C18-AR-II packed column
with linear gradient of acetonitrile.

According to this definition, 7 peptide fragments for PC were isolated

from ELN, OMP, and MLT, and their amino acid sequences were
determined by MALDI-TOF MS. As shown in Table 3, interestingly, all of
the isolated peptide candidates were obtained from the peptide
mixture produced by trypsin, while no significant differences in
peptide components before and after adsorption were observed from
those produced by chymotrypsin. These results suggested that
clustered hydrophobic amino acids recognized by chymotrypsin, such
as Phe, Tyr, Trp and others might interact strongly with the surface of
PC and PMMA. No positive result was obtained when the peptide
fragments produced from BIF were used. These results indicate that
there was no special motif of polypeptide with strong binding affinity
toward the surface of PC. Therefore, the fact that BIF itself was
preferentially adsorbed toward the surface of PC as shown in Fig. 2,
might be due to multipoint interaction of polypeptide chain of BIF that
was weakly interacted with the surface.

However, peptides with an affinity toward the PMMA surface were

isolated only from the peptide mixture of OMP with trypsin as shown
in Table 2. These results suggest that the binding affinities of peptide
fragments derived from ELN, MLT, and BIF were relatively low, probably
for the same reason as described above, while the surface of PMMA
(water contact angle: 78°) has a lower surface hydrophobicity than PC
(water contact angle: 88°), which may be more receptive to
protein/peptide adsorption.

Table 2. Identified peptide fragments binding to PC and PMMA.

Name Amino acid sequence Length pI

Candidates of PC-binding peptide

PC-MLT8 WTPIMSTVMEIGYDNVESQR 20 3.87

PC-MLT10 GDSDEWTFGAQMEIWW 16 2.88

PC-ELN8 QVGVPYIIVFLNK 13 9.49

PC-OMP6 NSNFFGLVDGLNFAVQYLGK 20 6.34

PC-OMP7 NILAVIVPALLVAGTANAAEIYNK 24 6.54

Candidates of PMMA-binding peptide candidates

PM-OMP19 SNGDGVGGSISYEYEGFGIVGAYGAADR 28 3.62

PM-OMP25 DVEGIGDVDLVNYFEVGATYTFNK 24 3.46

According to structural analysis of the parent affinity proteins, the