Biochemical Engineering Journal 18 (2004) 169–175

An alternating elution strategy for screening high affinity

peptides from a phage display peptide library
Haiqing Yu, Xiao-Yan Dong, Yan Sun∗
Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China
Received 17 March 2003; accepted after revision 26 August 2003

Abstract
An efficient procedure for the selection of high affinity clones from a heptapeptide phage display library was developed. Lysozyme
was used as a model protein to demonstrate the selection strategy. Effect of bovine serum albumin (BSA) concentration on screening the
phage library was discussed and proper BSA concentration on plate blocking was determined. The elution procedure was improved by
alternatingly eluting the bound phages with glycine–HCl buffer (pH 2.2) and high-concentration target protein solution. The modified
method was compared with others including the conventional protocol, and the results confirmed that the modified procedure could yield
high affinity phages that might be lost by other screening methods. Through comparison of the DNA sequences of foreign peptides of the
clones showing specificity to lysozyme molecules, the HWWW motif was found to be the necessary amino acid sequence for the affinity.
The electrostatic and hydrophobic interactions are considered to contribute to the affinity for the protein. Moreover, protein chromatography
with the immobilized HWWWPAS on Sepharose gel indicated the strong binding affinity of the peptide for lysozyme.
© 2003 Elsevier B.V. All rights reserved.
Keywords: Phage display library; Peptide; Ligand; Selection; Lysozyme; Affinity chromatography

1. Introduction Phage peptide libraries are often screened by two meth-

Affinity chromatography is one of the most effective
methods for the purification of proteins because affinity
interactions rely on the specific identification between the
desired protein and its ligand. The development of peptide
libraries has provided a relatively convenient and rapid way
for this purpose [1]. Peptide sequences can be easily dis-
played at the surface of filamentous phages, such as M13
and fd bacteriophages, by inserting corresponding oligonu-
cleotide sequences into the gene encoding for the minor
(pIII) or major (pVIII) surface protein [2]. Insertion of a
randomized nucleotide sequence of a given length can yield
phage-displayed peptide libraries, which constitutes useful
biochemical tools for the selection of peptides that specifi-
cally bind to a given target [1]. Many successful examples
prove the practicability of the technology. Many peptide
sequences that react with ligand such as antigens [3], mon-
oclonal antibodies [4], receptors [5] and proteins [6,7] are
isolated from phage display peptide libraries.
∗ Corresponding author. Tel.: +86-22-27404981;
fax: +86-22-27406590.
E-mail address: ysun@tju.edu.cn (Y. Sun).
ods; one needs the attending of solid phase, and the other is
carried out in liquid phase. In practical use, the former is em-
ployed more frequently. In this method, the desired protein is
attached on the surface of microtiter plates [8] or polystyrene
beads directly [9]. After the phage library is reacted with
the immobilized protein, the unbound phage particles are
washed away, and then the bound phages are eluted and
collected. The bound phages are amplified in Escherichia
coli cells prior to subsequent rounds of biopanning [10].
Usually, the phages that are specific to the target protein can
be enriched by three rounds of screening. Sometimes, how-
ever, the specific phages are obtained though six rounds or
more [11]. The gene extracted from the amplified phage can
be sequenced and the specific peptide sequence determined
[1].
When the bound phages are eluted with high or low
pH solutions in traditional methods, most of the selected
clones frequently show low to medium affinity for the target
[12,13]. Therefore, some improved procedures have been
suggested recently. For example, a stepwise decrease in pH
of the elution buffer during the final round of biopanning
was introduced by D’Mello and Howard [14], while Gaskin
et al. used the target protein solution as the elution buffer
[11], and Bruin et al. repeatedly eluted the bound phage

1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2003.08.006
170 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175
with triethylamine [15]. These changes are beneficial for
selecting high affinity clones.
In this article, we describe a screening procedure to further
improve the selection of high affinity binding clones from
a heptapeptide phage display library. Lysozyme was used
as a model to evaluate the procedure. The effect of bovine
serum albumin (BSA) concentration in the blocking buffer
on the selection of high affinity phages was first studied. In
the process of elution, low pH buffer and target protein so-
lution was used as the elution reagents in turn, and the high-
est specific clones were obtained in the last elution of each
round. We call this screening strategy an alternating elu-
tion procedure. The alternating elution method was applied
from the first round. By three-round screening, high affin-
ity clones were selected and the consentaneous sequences
were analyzed. Furthermore, the peptide with the highest
specificity to lysozyme was synthesized and immobilized to
Sepharose gel to observe its affinity binding property for the
protein.
2. Materials and methods
2.1. Reagents
The heptapeptide library and the E. coli 2738 were rece-
ived from New England BioLabs (Beverly, MA, USA).
Lysozyme (L 1096), BSA and 2,2" -azino-di-(3-ethylbenzthi-
azoline sulfonic acid) (ABTS) were purchased from Sigma
(St. Louis, MO, USA). The horseradish peroxidase labeled
mouse anti-M13 monoclonal antibody was obtained from
Amersham Biosciences (Buckinghamshire, UK). EAH
Sepharose 4B was purchased from Amersham Biosciences
(Uppsala, Sweden). Other chemicals of the highest grade
were received from local sources.
2.2. Microtiter plate coating and blocking
Lysozyme was adsorbed to the surface of wells of a
96-well polystyrene microtiter plate (Nunc, UK) as fol-
lows. Two hundred microliters of 0.1 mg/ml solution of
lysozyme in 0.1 M NaHCO3 (pH 8.6) was added to the
wells and incubated overnight at 4 ◦ C. After removing the
supernatant solution, 400 !l of, typically, 0.6% BSA (w/v)
in the NaHCO3 solution was added and incubation was
continued for 2 h at 4 ◦ C in order to block any remaining
binding sites before reaction with the phage library. The
wells were then washed six times with PBST (PBS buffer,
8 g NaCl, 0.2 g KCl, 1.44 g Na2 HPO4 and 0.24 g KH2 PO4
in 1 l, pH 7.4 adjusted with HCl, containing 0.05–0.2%
Tween 20).
2.3. Biopanning
For screening high affinity clones from the phage li-
brary, biopanning was performed by alternating elution of
the bound phages with glycine solution (pH 2.2) and tar-
get protein (lysozyme), different from those reported by
other researchers [4,14,16]. The alternating elution pro-
cedure was carried out as follows. In each round, about
1 × 1010 or 1 × 108 clones of the phage library were in-
cubated for 1 h with the immobilized lysozyme in PBS
containing Tween 20 (Tween 20 concentration was in-
creased gradually in the selections, 0.05% in the first round,
0.1% in the second round, and 0.2% in the third round).
The unbound phages were removed by washing the wells
10 times with the PBST. In elution step, the wells were
eluted four times and individual elution samples were col-
lected for further assays and/or phage amplification. The
elution in each round was conducted as follows: (1) 0.1 M
glycine–HCl buffer (pH 2.2) containing 1 mg/ml BSA was
added in the wells and incubated for 10 min; (2) 200 !l
of 0.2 mg/ml lysozyme solution was added and incubated
for 60 min; (3) the glycine–HCl buffer was added in the
wells again, incubating for 10 min; and (4) the final elution
was done by adding 200 !l of 0.2 mg/ml lysozyme solution
in the wells and incubating for 120 min. The phages col-
lected in the fourth elution were amplified for next round
biopanning.
2.4. ELISA methods
Enzyme-linked immunosorbent assay (ELISA) was car-
ried out using Maxisorb microtiter plates (Nunc). One
hundred microliters of 1 !g/ml solution of lysozyme in
0.1 M sodium carbonate buffer (pH 8.6) was first added
and the plates were incubated overnight at 4 ◦ C. Liquors
in the wells were discarded and the wells were blocked by
addition of 400 !l of 3% BSA (w/v) in PBS. After incu-
bation for 2 h at room temperature, the wells were washed
six times with the PBST. Phage samples were diluted as
necessary with the PBST, and 200 !l of each sample was
added to the well and allowed for adsorption for 1 h at room
temperature. After washing six times with the PBST, 200 !l
of 5000× dilution of the mouse anti-M13 MAb marked
with horseradish peroxidase was added and incubated at
room temperature for 1 h. After washing as before, 200 !l
ABTS solution (0.22 ng/ml ABTS in 0.05 M citric acid, pH
4.0; immediately before use, 36 !l of 30% H2 O2 solution
was added to 21 ml of the ABTS solution) was added and
incubated in the dark at room temperature for 15 min. The
plate was read at 405 nm using a model M550 microplate
reader (BIO-RAD, US). Control experiments were done in
parallel without coating lysozyme to the plates.
2.5. DNA sequencing
Purification of sequencing templates DNA from the se-
lected phases was carried out according to the instruction
of the manufacturer supplying the M13 phage library. DNA
sequencing was performed by Jingtai Company (Shanghai,
China).
 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175 171

2.6. Peptide synthesis and immobilization 0.3

The peptides were synthesized by solid phase method

by GL Biochem Company (Shanghai, China) using Fmoc
0.25

ELISA signal (405nm)

(9-fluorenylmethoxycarbonyl) chemistry [17]. The product
was purified by reversed-phase HPLC to a purity of 96%.
Peptide were immobilized on EAH Sepharose 4B by
the following method. Freeze-dried peptide powder was 0.2
dissolved in a coupling solution of 30% (v/v) ethylene gly-
col (pH 5.0) in distilled water to a final concentration of
6 mg/ml. The peptide solution was mixed with the drained 0.15
gel in a volume ratio of 2:1. The slurry was incubated for
5 h at room temperature. After peptide coupling, the unre-
acted amino groups on the gel were blocked by reacting
with 1 M acetic acid for 1 h at room temperature. Because 0.1
0.1 0.2 0.3 0.4 0.5 0.6 0.8 1
the N-terminus of the peptide had been Fmoc-protected,
BSA concentration (%)
it was deprotected at this stage using 20% (v/v) piperi-
dine in dimethylformamide for 10 min. Thereafter, the gel Fig. 1. Change of ELISA signal with BSA concentration for plate blocking.
was drained and washed six times alternatively with 0.1 M
Tris–HCl buffer (pH 8.3) and 0.1 M acetate buffer (pH 4.0) concentration for plate blocking. In separate experiments,
(each containing 0.5 M NaCl). The peptide coupling den- a row of wells were coated with lysozyme and blocked
sity was determined by reversed-phase HPLC analysis of with different concentrations of BSA. Then, approximately
the solution before and after coupling reaction. A Waters 1.3 × 1010 library phage clones were added to the wells
symmetry C18 reversed-phase column (Waters Corporation, and a process of ELISA was carried out. The results are
Milford, MT, USA) (4.6 mm i.d. × 150 mm, 5 !m particle shown in Fig. 1. As can be seen, BSA concentration higher
size) was used for the analysis. than 0.4% was needed to completely block the well surface.
Considering that the binding reaction between phages and
2.7. Affinity chromatography target protein molecules is possibly disturbed by using high
concentration of BSA, we employed 0.6% BSA solution for
Chromatographic experiments were performed on the plate blocking in the following experiments.
ÄKTA Explorer 100 System (Amersham Biosciences). The
eluted peaks were detected at 280 nm. One milliliter of the 3.2. Alternating elution procedure
gel coupled with the peptide was packed into an HR5/5
column (5 mm i.d., 5 cm length) (Amersham Biosciences). Alternating elution was applied in each round of biopan-
The column was washed and equilibrated with more than ning in order to preferentially and efficiently select high
20 column volumes (CVs) of 0.05 M Tris–HCl buffer (pH affinity clones. In every round, the phages in each elution
7.5) after a base line of the absorbance at 280 nm had been were collected and tittered to calculate recovery yield. The
reached. Then, 50 !l of 1 mg/ml lysozyme solution was results are listed in Table 1. In the first round, the titer of
injected and the column was washed with 10 CVs of the the collected phages decreased rapidly from the third elution
Tris buffer. The bound protein was eluted with the Tris
buffer containing 1 M NaCl or 0.1 M glycine–HCl buffer Table 1
(pH 2.2). After each experiment, the column was washed Yield of phages in each elution in three rounds and the total number and
alternatively with 0.1 M Tris–HCl buffer (pH 8.3) and 0.1 M recovery in each round
acetate buffer (pH 4.0) (each containing 0.5 M NaCl) and Round
re-equilibrated with 0.05 M Tris–HCl buffer (pH 7.5). The
1 (1.28 × 1010 )a 2 (1.62 × 108 )a 3 (1.12 × 108 )a
chromatographic experiments were performed at 0.2 ml/min
at room temperature. Yield (pfu/!l)
First elution 3.1 × 104 2.3 × 104 1.1 × 103
Second elution 3.5 × 104 8.5 × 103 3.9 × 103
Third elution 10 1.7 × 102 6.1 × 104
3. Results
Fourth elution 26 8.3 × 102 8.7 × 105

3.1. Determination of proper BSA concentration in Total 6.6 × 104 3.3 × 104 9.4 × 105
blocking buffer Recovery (%)b 5.16 × 10−4 0.023 1.06
aNumber of phages put in.
A series of BSA solutions of different concentrations in bRecovery is expressed as relative percentage of the total number of
blocking buffer were employed to find an appropriate BSA phage eluted compared to number of phage put in.
172 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175

0.5 Table 2
Yield of phages of the five elution methods (A–E)
Elution step Eluting agent Elution time (min) Titer (pfu/!l)
0.4
ELISA signal at 405nm

(A)
1 Glycine 10 3.4 × 104
0.3 (B)
1 Glycine 10 3.3 × 104
2 Glycine 10 7
0.2 3 Glycine 10 1
4 Lysozyme 60 1.8 × 102
(C)
0.1
1 Lysozyme 60 1.2 × 103
2 Lysozyme 60 0
3 Lysozyme 60 100
0 4 Lysozyme 60 7.2 × 102
1 2 3 4
Elution step (D)
1 Lysozyme 240 8.7 × 102
Fig. 2. ELISA signal values of the phages eluted from each step in the (E)
second round by the alternating elution procedure. 1 Glycine 10 3.1 × 104
2 Lysozyme 60 3.5 × 103
3 Glycine 10 15
step, from 3.5 × 104 to 26 pfu/!l. In the second round, the 4 Lysozyme 120 26
titer of phages collected from each elution also decreased,
but the decreasing trend became slow, from 2.3 × 104 to
8.3 × 102 pfu/!l. This is because the phages used for the
with only the glycine buffer (Table 2A), lysozyme solution
selection had been mostly high affinity clones obtained in
(Table 2C and D) or their simple combination (Table 2B).
the fourth elution of the first round. In the last round, the
The best elution effect was achieved by using glycine and
phenomenon was different from the first and second rounds.
lysozyme solutions in turn (Table 2E).
That is, the collected number of phages increased through
Furthermore, we compared the phages collected from the
the elutions. The number in the final elution was as high as
traditional method with those from the fourth elution by
8.7 × 105 . In addition, the total recovery increased rapidly
the alternating elution method in three rounds (Fig. 3). The
with the selection round.
specificity of the latter was higher than the former. After
The phages obtained from each elution in the second
three rounds, the ELISA signal of the phages from the tradi-
round were amplified and determined for ELISA assays. As
tional method was 0.374, but it reached 0.596 for the phages
shown in Fig. 2, the signal of the fourth-elution phages was
obtained by the alternating elution method.
the highest.

3.3. Comparison of alternating elution strategy with 0.6

other screening methods
Traditional
0.5
Alternating
ELISA signal at 405nm

Other four elution approaches were tested and compared

with the alternating elution method developed here. Method
0.4
A was a traditional one [10], by which the well was eluted
for 10 min with the glycine buffer. Method B was that the
well was eluted with the glycine buffer three times, each 0.3
time for 10 min, then the well was eluted with 0.2 mg/ml
lysozyme solution for 1 h. In method C, the well was eluted 0.2
with 0.2 mg/ml lysozyme solution four times, each time for
1 h. Method D was that the well was incubated for 4 h with 0.1
0.2 mg/ml lysozyme solution for only one time. In each
screening experiment, approximately 1.3 × 1010 library 0
phages were added to each coated and blocked well and the 1 2 3
clones remaining in the well after washing were collected Round
in each elution of the approaches to compare their yields
Fig. 3. Specificity of the phages collected from the traditional elution
(Table 2). As can be seen from the table, the alternating elu- method and the fourth elution by the alternating elution method. Empty
tion method (method E, see Table 2E) gave the highest total bars represent the phages collected by the traditional method. Shadow
yield. The bound phages could not be eluted completely bars indicate the phages obtained in the alternating elution method.
 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175 173

0.7

0.6

ELISA signal (405nm)

0.5

0.4

0.3

0.2

0.1

0
C 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Clone

Fig. 4. The specificity of the 24 clones randomly selected from the fourth elution in the third round screening. The shadow bars represent the clones
sequenced. The ELISA signal of library phages was compared as the negative control (C).

3.4. Amino acid sequences and relative affinities of the 0.8

phage peptides
P2
The specificity of the higher affinity clones was detected P14
ELISA signal at 405nm

by ELISA. By using the alternating elution strategy in each 0.6 P5

round of biopanning, the phages recovered from the fourth P16
elution in the third round were plated out to obtain individ-
ual clones for ELISA assays. All of the phages were selected 0.4
from plates with no more than 30 plaques. Twenty-four
clones were selected randomly and fourteen of them gave
signal values higher than 0.58 (Fig. 4). To obtain the gene
information of the specific clones, 7 clones were selected 0.2
randomly from the 14 high affinity clones for sequencing.
Six heptapeptide sequences of the seven clones were exactly
the same, HWWWPAS (His-Trp-Trp-Trp-Pro-Ala-Ser), and 0
the other one was HWTWWNL (His-Trp-Thr-Trp-Trp- 107 108 109 1010 1011
Asp-Leu). Two low affinity clones, P5 and P16 (Fig. 4), were Phage (pfu/ml)
also sequenced to observe their differences with the high
affinity clones. DNA and protein sequences of the clones are Fig. 5. Specific binding of the four phages displaying peptides, HWWW-
PAS, HWTWWNL, QNTLHPR, and VRIPVWH, to lysozyme.
listed in Table 3. The relative affinities of these sequences
are displayed in Fig. 5. These four peptides together with
their physicochemical properties are presented in Table 4.

Table 3
DNA and protein sequences of the clones selected from the third round

The insert segments are highlighted in bold. Amino acid was typed under the corresponding site with abbreviation.
a The sequence of other five high affinity clones was the same as P2.
174 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175

Table 4 procedure. Peak area analysis exhibits that the first peak
Some properties of the four peptidesa takes 19.9% of the total peak area. The small peak was
Peptide pI Molecular weight Hydropathicity washed out with 0.05 M Tris–HCl buffer at about 2 ml.
HWWWPAS 6.74 969.0 −0.929
This is considered to be the weakly bound component in
HWTWWNL 6.74 1042.1 −0.900 the lysozyme sample because reversed-phase HPLC anal-
QNTLHPR 9.76 864.9 −1.886 ysis showed that the lysozyme sample has only a purity of
VRIPVWH 9.73 906.1 0.386 about 86% [18]. Most of the protein was strongly bound
a Data from the website Expasy.org. to the column and could only be eluted with 1 M NaCl or
glycine–HCl buffer. The results indicate that the peptide
3.5. Affinity chromatography has a strong binding affinity to lysozyme.

The peptide HWWWPAS was synthesized and immobi-

4. Discussion
lized to Sepharose gel to observe its affinity binding ability
to lysozyme. The coupling density of the peptide was es-
Elution of bound phages is the key step in screening pro-
timated at 5.5 mg/ml. Fig. 6 shows the chromatograms of
tocol. The screening method by eluting phages bound to the
lysozyme. As can be seen, two peaks were eluted in the
target protein molecules that immobilized to the surface of
solid phase to obtain ligand-binding phages has been inves-
50 tigated by many researchers. Some protocols have proved
to screen phage display libraries efficiently [11,14]. Based
Absorbance at 280 nm (mAU)

on the methods reported in literature, we have improved the

40
elution procedure. By the alternating elution strategy, high
affinity clones can be readily selected. Other efforts that
30 aimed at improving the selection of high affinity clones,
such as extensive washing to dissociate low affinity phages
[13] and increasing the concentration of Tween 20 to de-
20 crease non-specific binding [19] were also incorporated into
our protocol.
1.0 M NaCl It should be noted that in the alternating elution process,
10 the target protein concentration was twice that for plate coat-
ing. After the first elution, quite a number of phages were
0 still bound in the well and a small quantity of bound phages
0 5 10 15 20 could still be eluted until the fourth elution (Table 1). It was
(a) Effluent volume (ml) confirmed that the phages eluted in the later stage had higher
affinity (Fig. 2). This is reasonable because the protonation
effect of the atoms near the affinity site, due to using low
50 pH buffer [20], is not enough to unbind the high affinity
phages. After elution with the glycine buffer to remove most
Absorbance at 280 nm (mAU)

40 of the low affinity clones, target protein solution of higher

concentration was added, which could dissociate high affin-
ity phages by competition with the coated target protein.
30 For those phages bound very tightly, the use of the glycine
buffer and target protein solution alternatingly could achieve
a satisfactory result. Results of the four control experiments
20
by other protocols illustrated that the alternating elution was
Gly-HCl necessary for screening high affinity clones (Table 2 and
10 Fig. 3). Here, we emphasize that the alternating elution be
applied from the first round to avoid the loss of high affinity
phages at the beginning of biopanning.
0 The insert sequences of the best six high affinity clones
0 5 10 15 20
showed complete consistency. The amino acid sequence
(b) Effluent volume (ml)
was HWWWPAS. The sequence of the other clone showed
Fig. 6. Lysozyme chromatography with Sepharose 4B coupled HWWW-
slightly lower specificity, HWTWWNL, appeared homol-
PAS as adsorbent: (a) elution with 0.05 M Tris–HCl buffer containing 1 M ogy to the former one (in italics). In contrast, those se-
NaCl (pH 7.5) after 10 CVs; (b) elution with 0.1 M glycine–HCl buffer quences of the next two clones presenting very low affinity,
(pH 2.2) after 10 CVs. QNTLHPR and VRIPVWH, showed non-homology to
 H. Yu et al. / Biochemical Engineering Journal 18 (2004) 169–175
HWWW sequence. So it is deduced that the amino acid
sequence HWWW give the main contribution to the affin-
ity for lysozyme. Moreover, the sequence HWWWPAS
and HWTWWNL are nearly the same in isoelectric points
and hydrophobicity (Table 4). They might set in a cer-
tain hydrophobic concave area on the surface of lysozyme
molecule. In addition, in the binding environment, pH 8.6,
lysozyme molecule (pI 11.2 [21]) is positively charged while
the two heptapeptides, HWWWPAS and HWTWWNL,
are negatively charged. So affinity binding by electrostatic
interactions can occur between the molecules. In contrast,
QNTLHPR and VRIPVWH as well as lysozyme are posi-
tively charged at pH 8.6, so little electrostatic attraction can
occur between the peptides and the protein.
The results of affinity chromatography indicate that the
peptide displayed on the phage coat can keep its affinity after
being synthesized and coupled to a solid matrix. Moreover,
the fact that the protein can be eluted by high-concentration
salt solution or low pH solution may also imply that the
electrostatic interactions are the main cause of lysozyme
binding to HWWWPAS. However, more experiments at ex-
tensive pH values and ionic strengths are needed to further
elucidate the binding mechanism.
In summary, the alternating elution strategy developed
here has been proved efficient for screening high affinity
peptides from a phage display library. It will be demon-
strated further by other target proteins and extensive chro-
matographic experiments.
Acknowledgements
This work was supported by the Natural Science Founda-
tion of China (No. 20025617).
References
[1] J.K. Scott, G.P. Smith, Searching for peptide ligands with an epitope
library, Science 249 (1990) 386–390.
[2] G.P. Smith, Filamentous fusion phage: novel expression vectors that
display cloned antigens on the virion surface, Science 228 (1985)
1315–1317.
[3] B. Wang, L.H. Ke, H. Jiang, C.Z. Li, P. Tien, Selection of a specific
peptide from a nona-peptide library for in vitro inhibition of grass
carp hemorrhage virus replication, Virus Res. 67 (2000) 119–125.
175
[4] G. Ferrières, S. Villard, M. Pugnière, J.C. Mani, N.T. Isabelle, F.
Rharbaoui, D. Laune, E. Loret, B. Pau, C. Granier, Affinity for
the cognate monoclonal antibody of synthetic peptides derived from
selection by phage display, Eur. J. Biochem. 267 (2000) 1819–1829.
[5] S.E. Cwirla, Peptide agonist of the thrombopoitetin receptor as potent
as the natural cytokine, Science 276 (1997) 1696–1699.
[6] J.J. Devlin, L.C. Panganiban, P.E. Devlin, Random peptide libraries:
a source of specific protein binding molecules, Science 249 (1990)
404–406.
[7] J. Qi, C.C. Zhou, H. Zhou, W. Li, B.Y. Liu, Y. Li, N. Fu, Searching
for the peptide ligands of interleukin-2 in phage-displayed peptide
library, Chin. J. Biochem. Mol. Biol. 15 (1999) 752–755.
[8] C.P. Hart, J.E. Martin, M.A. Reed, A.A. Keval, M.J. Prstelnik, J.P.
Northrop, D.V. Patel, J.R. Grove, Potent inhibitory ligands of the
GRB2 SH2 domain from recombinant peptide libraries, Cell. Signal.
11 (1999) 453–464.
[9] P.M. Keller, Identification of HIV vaccine candidate peptides by
screening random phage epitope libraries, Virology 193 (1993) 709–
716.
[10] J.F. Parmley, G.P. Smith, Antibody-selectable filamentous fd phage
vectors: affinity parification of target genes, Gene 73 (1988) 305–318.
[11] D.H. Gaskin, K. Starck, N.A. Turner, E.N. Vulfson, Phage display
combinatorial libraries of short peptides: ligand selection for protein
purification, Enzyme Microb. Technol. 28 (2001) 766–772.
[12] S.E. Cwirl, R.W. Barrett, W.J. Dower, Peptides on phage: a vast
library of peptides for identifying ligands, Proc. Natl. Acad. Sci.
U.S.A. 87 (1990) 6378–6382.
[13] R.W. Barrett, S.E. Cwirla, M.S. Ackernam, A.N. Olson, E.A. Peters,
W.J. Dower, Selective enrichment and characterization of high affinity
ligands from collections of random, Anal. Biochem. 204 (1992) 357–
364.
[14] F. D’Mello, C.R. Howard, An improved selection procedure for the
screening of phage display peptide libraries, J. Immunol. Methods
247 (2001) 191–203.
[15] R.D. Bruin, K. Spelt, J. Mol, R. Koes, F. Quattrocchio, Selection
of high-affinity phage antibodies from phage display libraries, Nat.
Biotechnol. 17 (1999) 397–399.
[16] H. Wu, G.N. Goud, M.R. Sierks, Artificial antibodies for affinity
chromatography of homologous proteins: application to blood clotting
proteins, Biotechnol. Prog. 14 (1998) 496–499.
[17] J.A. Buettner, C.A. Dadd, G.A. Baumbach, B.L. Masecar, D.J.
Hammond, Chemically derived peptide libraries: a new resin and
methodology for lead identification, Int. J. Peptide Protein Res. 47
(1996) 70–83.
[18] L.-Z. He, Y. Sun, Purification of lysozyme by multistage affinity
filtration, Bioprocess. Biosyst. Eng. 25 (2002) 155–164.
[19] D. McGregor, Selection of proteins and peptides from libraries
displayed on filamentous bacteriophages, Mol. Biotechnol. 6 (1996)
155–162.
[20] G.D. Liang, New Experimental Techniques for Molecular Biology,
Scientific Publishers Ltd., Beijing, 2001, pp. 101–128.
[21] A.L. Lehninger, Principles of Biochemistry, Worth Publishers, New
York, 1982, p. 128.