Research Article
pubs.acs.org/acscombsci
A High Performance Platform Based on cDNA Display for Efficient
Synthesis of Protein Fusions and Accelerated Directed Evolution
Mohammed Naimuddin*,†,‡ and Tai Kubo*,†,§
†
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Central 6, 1-1-1 Higashi, Tsukuba,
Ibaraki 305-8566, Japan
‡
Janusys Corporation, #508, Saitama Industrial Technology Center, Skip City, 3-12-18 Kami-Aoki, Kawaguchi, Saitama 333-0844,
Japan
§
Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST),
2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan
S Supporting Information
*
ABSTRACT: We describe a high performance platform based
on cDNA display technology by developing a new modified
puromycin linker-oligonucleotide. The linker consists of four
major characteristics: a “ligation site” for hybridization and
ligation of mRNA by T4 RNA ligase, a “puromycin arm” for
covalent linkage of the protein, a “polyadenosine site” for a
longer puromycin arm and purification of protein fusions
(optional) using oligo-dT matrices, and a “reverse transcription
site” for the formation of stable cDNA protein fusions whose
cDNA is covalently linked to its encoded protein. The linker
was synthesized by a novel branching strategy and provided >8-fold higher yield than previous linkers. This linker enables rapid
and highly efficient ligation of mRNA (>90%) and synthesis of protein fusions (∼50−95%) in various cell-free expression
systems. Overall, this new cDNA display method provides 10−200 fold higher end-usage fusions than previous methods and
benefits higher diversity libraries crucial for directed protein/peptide evolution. With the increased efficiency, this system was
able to reduce the time for one selection cycle to <8 h and is potentially amenable to high-throughput systems. We demonstrate
the efficiency of this system for higher throughput selections of various biomolecular interactions and achieved 30−40-fold
enrichment per selection cycle. Furthermore, a 4-fold higher enrichment of Flag-tag was obtained from a doped mixture
compared with that of the previous cDNA display method. A three-finger protein library was evolved to isolate superior
nanomolar range binding candidates for vascular endothelial growth factor. This method is expected to provide a beneficial
impact to accelerated drug discovery and proteome analysis.
KEYWORDS: directed evolution, protein fusions, cDNA display, puromycin linker, high throughput, drug discovery
■
INTRODUCTION
display is generally accomplished in approximately 2−3
weeks,1,4 whereas it can span 5−8 weeks for the complete
process involving ∼10 cycles of selection for others.12−14 In
vitro selection and evolution of nucleic acids has been
successfully automated for the discovery of novel ligands.15
However, the display of proteins is difficult to be adopted by
the automated systems due to problems in coupling of the
downstream procedures. For instance, the requirement of the
cellular phase in the case of cell-dependent methods, such as
phage display, yeast display, and others, is an impediment to the
coupling process. Nevertheless, phage display has been
successfully used in semiautomated and automated approaches.16,17 In principle, cell-free translation methods are
potentially capable of automation; however, there are no
State-of-the-art display technologies are powerful methods that
have contributed to the development of novel biomolecules for
various purposes, including research tools, diagnostic reagents,
and drug discovery.1−3 Phage display has been a frontrunner
among various available methods4 and has been successfully
employed for various purposes.5−7 However, fundamentally
cell-free methods provide certain advantages over cell-dependent methods, such as higher molecular diversity, flexibility for
incorporation of unnatural residues into proteins/peptides, and
post-translational modifications such as disulfide shuffling
reactions.8−10
Despite the advantageous properties and advances,11 these
methods have not gained general applications as phage display.1
Plausible reasons could be easier and robust linking of genotype
to protein and established and simplified procedures in phage
display compared with ribosome, mRNA, and cDNA display.
The complete process of selection/evolution using phage
© 2016 American Chemical Society
Received: September 2, 2015
Revised: January 20, 2016
Published: January 26, 2016
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Figure 1. Design and characteristics of the new puromycin linker-oligonucleotide and efficiency of template preparation for cDNA display. (a)
Schematic of the designed linker-oligonucleotide molecule. Linker-N contains four functional features: a ligation site for Y-ligation by T4 RNA ligase,
a puromycin moiety for the covalent linking of the synthesized protein, poly-A for longer puromycin arm and purification on oligo-dT resin, and a
priming site for reverse transcription to synthesize cDNA (branching of RT site was done via Me-dC; see Experimental Section). (b) Schematic for
the formation of mRNA protein fusion (1) and cDNA protein fusion (cDNA display) (2) are shown. The synthesized protein fusion (containing
mRNA) is linked to cDNA after reverse transcription synthesis of cDNA. (c) Genetic constructs adopted for cDNA display. SP6 denotes SP6
promoter and cap site; UTR, 5′ untranslated region of Xenopus β-globin; Kozak sequence; ATG, initiation codon; PDO/BDA/3F/6R14 are the
genes; Spc, spacer GGGS (G = glycine; S = serine); His-tag for IMAC purification; and Y-tag contains sequence for the docking of linkeroligonucleotide on mRNA. (d) Time-response reactions using PDO mRNA and puromycin linker-oligonucleotide (ratio 1:1) were performed;
equivalent moles were electrophoresed and analyzed by fluorescence detection (left panel) and with Sybr Green for all nucleic acid components
(right panel). M denotes size marker; linker, puromycin linker-oligonucleotide; 10 min, ligation reaction performed for 10 min; mRNA-linker,
mRNA ligated to linker-oligonucleotide; linker (RNase H), linker-oligonucleotide obtained after digestion of mRNA-linker with RNase H. Other
representative examples used in this study (3F, 6R14, BDA) are shown in (e) and quantified (f). Error bars were calculated from three independent
experiments.
reports of successful automated high-throughput platforms.
Semiautomated selections based on microfluidics have been
successful.18 This is mainly attributable to the procedures
involved in these technologies that are difficult to be optimized,
such as preparations of template mRNA conjugates and protein
fusions, and are suited more for manual operations that have
achieved novel and superior ligands.12−14,18 Higher throughput
and exponential generation of novel ligands and reagents are
highly desirable for large scale projects (such as proteome
mapping) and accelerated drug discovery.1
In this paper, we describe the optimization of the precursor
and successive procedures required for the synthesis of protein
fusions to alleviate execution time and simplify the process
toward an efficient system. On the basis of our findings, we
present a new cDNA display method by designing a new
modified puromycin linker-oligonucleotide that enables rapid
and efficient generation of templates and synthesis of protein
fusions and significantly reduces the time required for one
selection cycle to less than 8 h. The application of the method
for parallel and multiple selections was evaluated using various
test biomolecules for known targets (immunoglobulin,
acetylcholine receptor, and interleukin-6 receptor) and was
demonstrated to provide higher throughput. We also
demonstrate higher selection efficiency using a doped mixture
of ligand and library for targets such as antiflag antibody.
Furthermore, we show improved screening and higher affinities
obtained with this system compared with the previous report
using a three-finger library panned for the target vascular
endothelial growth factor (VEGF). Other potential advantages
and applications are also discussed.
■
RESULTS AND DISCUSSION
Characteristics of the New Puromycin Linker-Oligonucleotide. Preparation of templates for subsequent downstream steps in the case of mRNA/cDNA display is a complex
process owing to the yields that have been either low or require
prolonged time to increase the efficiency of the system. In the
case of decreased efficiency, incorporation of purification steps
is required for the recovery of the specific product and
elimination of the reactants followed by the detection of
purification efficiency. In general, purification steps inherently
bring further reduced yields in addition to the increased
execution time and the difficulty to be adopted by automated
high-throughput systems. For these problems to be overcome,
the design of the puromycin linker-oligonucleotide, which is
central to cDNA (or mRNA) display, is important. We have
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beneficial to large-scale projects, e.g., for proteome mapping. A
schematic of the formation of mRNA protein fusions is shown
in Figure 1b (1). The protein fusions are linked to cDNA after
complementary strand synthesis by reverse transcription and
provide stability to the fusions (Figure 1b (2)).
Ligation Efficiency of the New Linker-Oligonucleotide. An important initial step in cDNA display (or mRNA
display) is the ligation of mRNA to puromycin linkeroligonucleotide. Therefore, we evaluated the efficiency of
ligation and ligation time with our newly designed molecule.
For this purpose, we used Pou-binding domain of Oct-1
(PDO), B domain of protein A (BDA), three-finger (3F), and a
ligand of interleukin-6 receptor (6R14).20,22,24 Using linker-N
and the constructs (Figure 1a and Figure 1c), mRNAs of
different sizes and compositions were found to be rapidly
ligated at an equimolar ratio in just 10 min and attained 90−
95% efficiency (Figure 1d−f). Incubation for longer time did
not increase the ligation efficiency significantly. Ligation was
confirmed by digestion of the mRNA-linker conjugates with
RNaseH that cleaves the mRNA hybrids formed with the
complementary sequence of linker-N. The complementary
sequence is designed to facilitate the docking of linkeroligonucleotide on mRNA and provide stability during the
ligation reaction.13,20,21,24 The design of this simplified ligation
site that contains perfectly aligned complementary sequences
and is free from the formation of secondary structures may have
facilitated the improved ligation efficiency. In “puro-linker”, the
ligation site contains complicated secondary structures in the
“biotin loop” (to form the self-complementary restriction site
for PvuII), which may require prolonged time to stabilize and
achieve higher ligation.13,20
Several strategies for ligation have been previously reported,
namely, splint ligation, psoralen cross-linking, and Yligation10,12−14,18,20,21,24−27 (Table 2). Factors such as
stoichiometry of the reactants (i.e., mRNA, splint DNA, and
puromycin linker-oligonucleotide), incubation time, and
ligation efficiency are important in determining the total
execution time. The splint ligation method requires a longer
execution time owing to the slow ligation reaction, low ligation
efficiency, and gel purification steps required to purify ligated
mRNA from the unused mRNA.10,12,14 Psolaren photo-crosslinking is extremely efficient (performed at the molar ratio of
1:2 for mRNA:linker) and fast;25 however, the use of UV
irradiation may cause damage to the mRNA and requires
removal of excess linker. Y-ligation by enzymatic action of T4
RNA ligase is also very efficient; however, the reported time of
ligation and the stoichiometry of mRNA and linkeroligonucleotide have varied.13,20,26 Linker-oligonucleotide in
excess of 2−200 fold to mRNA requiring a ligation time of 1−
40 h have been used.13,26 The extended ligation time is not
beneficial to mRNA owing to its labile nature, and the use of
excess linker is not a cost-effective approach. Our ligation
report with linker-N has successfully dealt with ligationassociated bottlenecks by rapidly generating mRNA-linker
conjugates without the need for purification in a highly costeffective manner (10 min ligation at an equimolar ratio of
mRNA and the expensive linker-oligonucleotide) that can be
directly used for translation and preparation of protein fusions
(Table 2). Thus, this mRNA-friendly ligation method is
potentially capable of smooth incorporation to automated
high-throughput platforms. A recent report showed a reduced
ligation time of 10 min by modification of the “puro-linker” and
introducing a ribonuclease T1 site.21 Linker-N is free from the
designed a new modified linker-oligonucleotide (linker-N) to
increase the efficiency of the system.
Linker-N consists of four major portions. First, a “ligation
site” for the docking of mRNA by hybridization with the linker
and then ligation by T4 RNA ligase.19 Second, a “puromycin
arm” for the covalent linking of puromycin and the nascent
protein on a ribosome. Third, a “polyadenosine site” for a
longer puromycin arm and purification of the conjugates from
the lysate. Fourth, a “reverse transcription primer site” for the
conversion of labile mRNA to stable cDNA protein fusions,
whose cDNA is covalently linked to its encoded protein (Figure
1a). The formation of mRNA/cDNA hybrids provides stability
to the mRNA, prevents degradation of mRNA, and restricts the
formation of secondary structures that may act as aptamers.
In the recent past, linkers with the features mentioned above
have been designed and published.13,20,21 The modified
distinctive features of linker-N first include a simplified ligation
site containing complementary sequences that is free of
complex secondary structures designed to include the biotin
binding site and restriction cleavage site reported earlier.13,20
Second, the puromycin arm has been elongated by calculating
the physical dimensions of the eukaryotic ribosomes and adding
18 adenosine residues (oligo-dA) with the rationale of
generating higher fusions by close proximity to the tunnel of
stalled ribosomes. In addition, the oligo-dA site can be utilized
for purification from the translation lysates, in particular, rabbit
reticulocyte lysate where His-tag purification cannot be used. It
may be noted that to increase the length of the puromycin arm
any sequence is expected to work fine. We have chosen oligodA (which is also used for oligo-dT purification in mRNA
display) to have dual function, i.e., increase the length of the
puromycin arm and use for purification purposes via oligo-dT,
if required. Third, the reverse transcription site is a bit longer
and contains five bases with higher GC content to provide
stability to the complementary bases.
The process of the synthesis of linker-N has been optimized
on a synthesizer by a novel strategy to branch the reverse
priming site by the use of 5-Me-dC (5′-dimethoxytrityl-N4-(Olevulinyl-6-oxyhexyl)-5-methyl-2′-deoxycytidine (see Experimental Section). The final yields were more than 8-fold higher
than those achieved by the manual chemical coupling method
that utilizes EMCS, [N-(6-maleimidocaproyloxy) succinimide],
to link the two modified oligonucleotides to form “puro-linker”
or “SBP linker” in the cDNA display13,21 (Table 1). The
Table 1. Comparison of the Synthesis of Puromycin Linkers
for cDNA Display
linker type
coupling
chemical type
coupling
chemical
linker-Na
puro-linkerb
monofunctional
bifunctional
(5-Me-dC)c
EMCSd
side chain
(branch)
synthesizer
manual
coupling
final
yield
4%
≤0.5%
a
This paper. bYamaguchi et al.13 c5′-Dimethoxytrityl-N4-(O-levulinyl6-oxyhexyl)-5-methyl-2′-deoxycytidine. d[N-(6-Maleimidocaproyloxy)
succinimide].
introduction of the synthesizer in the synthesis of linker in its
final form eliminates human variations, reduces time and cost
by more than 10 times, and greatly enhances the reproducibility
of the process, including the downstream processes. A
conservative estimation suggests that the amount of linker-N
synthesized in a single process (∼40 nmoles) will be sufficient
for ∼80 selections consisting of 10 rounds per selection may be
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Table 2. Comparison of the Efficiencies of Various Puromycin Linker-Oligonucleotides
linker type
linker-mRNA ratio
ligase used
ligation time
efficiency
purification of mRNA-linker
protein fusion
RRL
WGE
final fusion yield (% of input)
this paper
cDNA displaya
cDNA displayb
mRNA displayc
mRNA displayd
1:1
T4 RNA ligase
10 min
>95
not required
1:1−1:1.5
T4 RNA ligase
10 min
>95
not required
1:1−1:4
T4 RNA ligase
60 min
>90
required
1:200
T4 RNA ligase
2400 min
80%
required
1:1
DNA ligase
60 min
20%
required
≤65%
≤95%
∼60%
≤30%
≤30%
1%e
≤20%
ND
0.2%
≤10%
≤70%
∼10%
≤40%
ND
∼6.5%
a
Mochizuki et al.21 and Ueno et al.31 bYamaguchi et al.13 and Naimuddin et al.20 cMiyamoto-Sato et al.;26 dLiu et al.12 and Takahashi et al.14 eThis
value represents the yield before purification via the C-terminal tags. The final fusion yield was not reported.
Figure 2. Formation of protein fusions in different cell-free lysates and purification. (a) Three linkers were prepared to test the efficiency for the
formation of protein fusions in two lysates. These linkers differ in the repeats of adenosine residues. Linker-N contains 18 A, linker-N-10A contains
10 A, and linker-N-0A contains zero A residues. (b) Protein fusions in rabbit reticulocyte lysate. Proteins that form covalent fusion with puromycin
are detected as a shifted band (indicated as mRNA-linker-protein) compared to mRNA-linker band of lower MW. Efficiency of fusion formation
using the three linker-oligonucleotides is shown in the graph “RRL”. (c) Protein fusions in wheat germ lysate. Translation was performed for 10 min
followed by incubation with high salts for 30 min to 1 h (see Experimental Section for details) using the four templates (PDO, BDA, 3F, and 6R14);
0 denotes the fraction sampled after 10 min of translation; 1, fraction incubated for 1 h without high salts; 2, fraction incubated for 30 min in high
salts, and 3, for 1 h. (d) Performance of the fusion formation of other linker-oligonucleotides using the four templates. The left panel is for linker-N10A and the right panel for linker-N-0A. Fusion formation was performed for 1 h, and % fusion formation with the three linkers is shown in the
graph indicating “WGE”. (e) One-step purification of His-tagged protein fusions by IMAC for separation from the untranslated mRNA-linker. F.T.,
flowthrough; E1, eluate 1; and E2, eluate 2. All fractions (F.T.−E2) were reverse transcribed and digested with RNase H to remove mRNA. PDO
correspond to mRNA-protein-linker. cDNA-protein-linker bands migrate faster than mRNA-protein-linker. The bands have been adjusted from the
same gel to show the purification of protein fusions. Error bars were calculated from three independent experiments.
use of ribonuclease that might create problems for controlling
contamination for mRNA, although it is used after reverse
transcription.
cDNA Display. Translation and maturation into protein
fusions is another important area that needs attention as this
directly determines the library size and can influence downstream purification processes. In various cell-free lysates, the
template mRNA-(linker-N) conjugates of PDO, BDA, 3F, and
6R14 were able to generate protein fusions very efficiently.
Fusion formation was achieved at 50−65% in rabbit
reticulocyte lysate (RRL) (Figure 2b), and 85−95% fusion
formation was observed in wheat germ lysate (WGE) in all four
templates tested (Figure 2c). The presence of high salts (MgCl2
and KCl) for fusion maturation was an essential requirement
for both lysates tested. In WGE, ∼20−30% fusion formation
was observed in the absence of salts for an incubation time of 1
h (lane ‘1’, Figure 2c). However, an increase in fusion yields
was evident with the inclusion of high salts and incubation for
30 min to 1 h (lanes ‘2’ and ‘3’). In general, even 30 min for
maturation in high salts was sufficient to achieve 50−70%
fusions. The intactness of mRNA-linker and mRNA-proteinlinker was calculated by the additive values of the individual
band intensities and compared with the mRNA-linker of lane
‘0’. A benchmark of 0.9 (absolute value of 1 for lane ‘0’, i.e.,
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Figure 3. Schematic for the formation of protein fusions with different linkers. (A) Translation (without linkers). Proteins are synthesized by
ribosomes by reading out the mRNA framework. Ribosomes of eukaryotic origin have an average height of 25−30 nm. (b) Protein fusion with purolinker/SBP/L-N-0A. The short linkers (∼10−20 nts; 7 nm long) have been shown with respect to the length of ribosomes (assuming 15−20 nm of
length from mRNA). Spacer is shown in orange, and puromycin (red) is at the top. The puromycin forms a covalent bond with the protein at the Psite of the ribosome and releases the protein. (c) Protein fusion with linker-N. Formation of fusion with the longer puromycin arm of linker-N (∼40
nts; 12−14 nm) consisting of oligo-dA (shown in green). (d) Depending on the position of ribosomes in solution after protein synthesis, linker-N
with the oligo-dA “stem” and flexible spacers may provide the required flexibility to form a structure that may increase the probability of the
formation of more fusions.
apparent difference observed between RRL and WGE.31
However, in an earlier report, higher efficiency of fusion
formation (∼70%) was reported for WGE.26 Thus, the design
of linker is important in that it can play a critical role in the
efficiency of protein fusion formation plausibly based on the
exploitation of ribosome concentration that is responsible for
protein synthesis.
Next, we investigated the effect of the length of the
puromycin arm by modulating the number of adenosine
residues. We constructed two more linkers: one that contained
no adenosine residues (L-N-0A) and another that contained
ten adenosine residues (L-N-10A) (Figure 2a). The formation
of protein fusions in RRL was found to be ∼20% (L-N-0A) and
∼30% (L-N-10A) (Figure 2b). In WGE, fusion formation was
∼30% (L-N-0A) and ∼60% (L-N-10A) (Figure 2d). These
experiments indicate that the number of repeats of adenosine
plays a role in the formation of protein fusions and directly
correlates with the difference in the efficiencies of the
production of protein fusions. Furthermore, given the constant
concentration of ribosomes in WGE, the three different linkers
tested here have generated varying amounts of protein fusions.
These results emphasize that the design of linker is important
to probe the efficiencies of production of protein fusions. It
may be noted that L-N-0A, which is similar to puro-linker,13
and SBP,21,31 as these linkers are devoid of oligo-dA, provided
consistent efficiency of protein fusion formation (20−30%) in
the two lysates.31 By considering the physical parameter of the
linkers (with/without oligo-dA) and the ribosome, the
difference in the efficiencies of the linkers may be partially
explained. Eukaryotic ribosomes have an average height of 25−
mRNA-linker) was set, which indicates 90% of the mRNAlinkers and mRNA-protein-linkers were not degraded. Most
lanes were in line with the benchmark value. Because of the
presence of a fluorescein moiety for detection, some artifacts
may be observed as FITC is influenced by several factors, such
as exposure to light, ionic strength of buffers, and so forth.
Other fluorescent moieties, such as Alexa and others that are
not easily influenced by the environment, including buffers,
may be used in future. We reasoned that the design of a longer
puromycin linker arm (i.e., inclusion of repeats of adenosine
residues) may have contributed to the higher yields achieved
here.25 Several papers have been published that report the
synthesis of ∼5−40% protein fusions in RRL12−14,25 and ∼70%
in WGE using a single template.26 Our results show templateindependent higher fusion yields with various templates (Figure
2; Table 2). The difference in the fusion formation with two
different lysates can be partially explained based on the number
of ribosomes that are more than one order higher in WGE (2.5
× 1015 per ml or 4 μM) compared with RRL (1.2 × 1014 per ml
or 0.2 μM).8,28−30 The ribosome and mRNA-linker ratio is
1:1−2:1 (0.1−0.2 μM ribosome and 0.1 μM mRNA-linker) in
the case of RRL. The ratio is approximately 40:1 in the case of
WGE. The near equimolar ratio in RRL may not be sufficient
for higher fusion yields given the “single-turnover” in display
technologies due to the absence of a stop codon.10−14 On the
other hand, the high ratio in WGE may provide sufficient
ribosomes for higher fusion yields obtained here.
It is noteworthy that linker-N was also efficient in providing
the distinction between the efficiencies of the two translation
systems (RRL and WGE). In a recent report, there was no
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Figure 4. cDNA display with linker-N and its demonstration for higher throughput of in vitro selection. (a) Schematic of the steps essential for
performing cDNA display using wheat germ lysate. DNAs generated by PCR are transcribed to mRNAs, which are purified and quantified. mRNAs
are then rapidly ligated to linker-N, translated, and matured to display proteins in high salt conditions. Purification is achieved by Ni-NTA magnetic
beads, and cDNAs are synthesized on beads, eluted, and subjected to selection against the targets that are immobilized on streptavidin (SA) matrix.
The selected/binding molecules are eluted and amplified for analysis. The schematic for selection using PDO/BDA mixture screened for IgG is
shown. The time required for each step is indicated in green boxes, and the whole process can be completed in 6−8 h. (b) Selection and analysis by
PCR. Four model proteins were prepared, and three parallel selections were performed according to the steps outlined in (a). Mixtures of a
nonbinder and a binder (ratio of 20:1) were translated, processed, and subjected to selection against various targets (1−3). (1), IgG that binds to
BDA; (2), AChBP that binds to 3F, and (3), IL6-R whose ligand is 6R14. Selection efficiency was monitored by the change in the ratio of “before”
and “after” selection fractions and expressed as the “number of fold enrichment” shown in (c). Error bars were calculated from three independent
experiments.
30 nm (29.4 nm for wheatgerm) and an average width of 27−
31 nm.32,33 Linker-N, which is approximately 40 nucleotides,
has a length of 12−14 nm, and puro-linker/SBP/linker-N-0A is
approximately 7 nm long (∼10−20 nts).31 Linker-N with a
longer puromycin arm is geometrically better positioned to
form protein fusions compared with other short linkers by
approaching the tunnel of ribosome (assuming that the length
of the ribosome is approximately 15−20 nm from mRNA out
of the height of 29.4 nm for wheatgerm), as shown
schematically in Figure 3a−c. On the basis of these
observations, we present a model where the “stem” portion
(polyadenosine) provides the required length and presumably
stability to the flexible spacers (assuming oligo-dA has a rigid
DNA structure) and increases the efficiency of the formation of
protein fusions (Figure 3d); however, the stability provided by
oligo-dA requires further experimental investigation.
For selection experiments, synthesis of cDNA has been
suggested to be beneficial to reduce mRNA degradation and
formation of secondary structures of mRNA, especially when
libraries are used for selection.12−14,20,27 For performing reverse
transcription, the protein fusions require purification for
separation from the lysate and also to remove the stalled
ribosomes (due to the absence of a stop codon). Furthermore,
protein fusions also need to be purified utilizing the C-terminal
tags to isolate full-length protein by eliminating proteins
generated by premature termination codons in libraries. In
general, two-step purification has been used in mRNA and
cDNA displays. The incorporation of multiple purification steps
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Figure 5. Selection of the complex mixture doped with Flag-tag. (a) Constructs adopted for the preparation of the complex mixture. The Flag-tag
construct contained the residues of Flag-tag (DYKDDDDK) necessary for its binding to the anti-Flag M2 antibody flanked by the components
required for cDNA display with linker-N. The 8-mer library contained random residues flanked by components for cDNA display. (b) Selection
cycle. A complex mixture containing 8-mer library (nonbinder) was doped with 0.1% Flag-tag (binder) and subjected to selection cycle procedures of
transcription, ligation to linker-N, translation in WGE to synthesize mRNA fusion proteins, purification utilizing the His-tag and reverse transcription
to synthesize cDNA fusion proteins, selection on anti-Flag M2 antibody matrix, and PCR amplification. The amplified product undergoes the same
process for 2 additional cycles and is then subjected to cloning and sequencing of the clones. The same complex mixture was also subjected to
selection using procedures with “puro-linker”.13 (c) Summary of the selection result. The sequences of the clones of the complex mixture were
obtained at R0 (prior to selection) and R3 (after 3 selection cycles) for both linkers. The % population indicates the fraction of Flag-tag sequences in
the total number of sequences analyzed (given in parentheses).
often results in reduced yields in the range of 0.2−6.5%.12−14
Because of the formation of higher protein fusions, we have
used gentle one-step IMAC purification via His-tag and
performed reverse transcription. An estimated 60−80% of
purified cDNA protein fusions were found to be isolated from
lysates in less than 30 min (Figure 2e). Thus, taking into
consideration all the steps starting from fusion formation to
end-usage purified cDNA fusions, our method provides
approximately ∼10−200 fold higher fusions than previously
reported methods (Table 2).12,13 Higher yields of protein
fusions will directly influence the diversity of proteins and
increase the landscape of available proteins that may benefit in
vitro selection and evolution of proteins/peptides.34 Furthermore, the reduction of steps, minimizing purifications, and the
utilization of magnetic matrices makes this system potentially
amenable and friendly to high-throughput platforms.
In Vitro Selection. The cDNA display proteins synthesized
with linker-N were evaluated for performance in affinity
selection experiments using various known targets according
to the process outlined in Figure 4a. The whole process of one
cycle of selection can be completed in an accelerated manner
requiring 6−8 h. Two ligands that do not contain disulfide
linkages (PDO and BDA)22,23 and two ligands that require
disulfide linkages for the folded structure to maintain their
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Figure 6. Analysis of the selection of the 3F library for VEGF. (a) The three-finger scaffold contains three fingers protruding from the globular head,
and this topology is provided by the four disulfide linkages (shown in green). The randomization of the tips of loops facilitates preparation of library
(shown by fluorescent green loops). The genetic construct for the preparation of the library is shown below. (b) Analysis of the R7 library by binding
assays. The 3F library (cDNA fusions) after seven rounds of selection (R7) was subjected to a binding assay to evaluate its potential. The three
fractions were allowed to bind to (1) streptavidin beads (SA; matrix negative control), (2) VEGF beads, and (4) Fc beads (protein negative control).
The fourth fraction was the reverse transcribed R7 library (without fusion) to evaluate binding via the nucleic acid portion (3). The eluted fractions
were PCR amplified and analyzed by gel electrophoresis. The left panel represents a schematic of the assay, and the right panel contains the gel
electrophoresis profiles. (c) The three fractions, (1), (2), and (4), were subjected to the binding assay and detected using ELISA (see Experimental
Section). (d) Summary of the sequences and affinities of clones. The sequences were divided into three groups based on the frequency of occurrence
(given in parentheses). The affinities were measured by ELISA. Error bars were calculated from three independent experiments.
function (3F and 6R14, derived from a 3F library)20 were
chosen for selection. PDO binds to DNA (OBD) and was
chosen as a nonbinder to assess the selection efficiency. BDA
binds to IgG,22 3F binds to AChBP, and 6R14 has affinity for
IL-6R.20 The efficiency of selection was expressed as number of
fold enrichment and found to be 30−40 for the three ligand/
receptor pairs after a single selection cycle (Figure 4b and c).
These values are comparable to or better than those in previous
reports.10,13 Thus, protein ligands (both disulfide-deficient and
-rich) synthesized by the cDNA display method with linker-N
can bind to diverse targets, such as macroprotein (IgG), soluble
channel receptor (AChBP), and soluble signal transducing
receptor (IL-6R).
Selection from Complex Mixtures. An important aspect
and final goal of display technologies is to select desired
candidates (e.g., for binding) from undesirable (nonbinding)
candidates contained in a library of molecules. We evaluated
the performance of our method using (a) a doped mixture of
known ligand (binder) and library molecules (nonbinder) for
the natural target and (b) totally random libraries for a given
target.
We chose Flag-tag (known ligand; binder) for the purpose of
preparing doped mixtures with a library of 8-mer peptide library
(nonbinder) mixed at a ratio of 1:1000 (Flag-tag: library; or
0.1% Flag-tag) (Figure 5a). Please note that any nonbinding
peptide of comparable size can be used for this experiment. For
comparison, the whole process was also performed with “purolinker”.13 Selection was carried out against immobilized antiFlag M2 antibody (natural target for Flag-tag) for three rounds
simultaneously with both methods (Figure 5b). After selection,
the pools were cloned and sequenced. The flag-tag sequences
were 80% (16 of 20 clones) after 3 rounds of selection that
utilized linker-N up from 0.1% compared with 20% that were
observed with “puro-linker” (Figure 5c; see Supporting Table
1). It may be noted that similar results (17% sequences) were
obtained with “puro-linker” in the previous report.13 These
results demonstrate that, with linker-N, improved enrichment
(4-fold) of Flag-tag sequences for its natural target, anti-Flag
M2 antibody, was obtained relative to that of “puro-linker”. We
speculate that, in the case of linker-N, the increased formation
of protein fusions with a reduced “leftover” template mRNAlinker has facilitated improved selection. The leftover mRNAlinker is significantly higher in the case of “puro-linker” (∼80%)
compared with the protein fusions (∼20%)13,20,21 and is
difficult to purify completely even after utilizing protein
purification via his-tag. The untranslated mRNA-linker
converted cDNA-linker can potentially reduce the S/N ratio
during PCR amplification by increasing the “noise” (i.e.,
unwanted/nonbinder DNA species) and interfere with the
outcome of the selection process.
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Table 3. Comparison of Performance of Puromycin Linkers for cDNA Display
a
linker type
library/ml
library used
selection rounds
major group
highest affinity (Kd)
linker-Na
puro-linkerb
6 × 1012
4 × 1010
3 × 1012
1.2 × 1011
7 (7 days)
10 (30 days)
60%
70%
2.1 ± 0.5
48 ± 8
This paper. bNaimuddin et al.20
process. Another interesting result was observed in the highest
affinity molecule generated in each method, which is higher by
approximately 1 order of magnitude obtained with linker-N
(2.1 vs 48 nM); affinities varied by approximately 2 orders of
magnitude of the major population generated in each method
(2.1 vs 152 nM).20 This is consistent with the theory that
selection outcome is dependent on the diversity of the initial
library; however, this needs more investigation.28,34 These
results demonstrate the advantages that can be useful for cDNA
display by employing linker-N to obtain superior candidates at
far lesser cost and labor in an accelerated manner.
VEGF binding molecules have been isolated from antibody
and cysteine constrained libraries by using phage display with
affinities in the range of 1.8−470 nM.35,36 The candidates
generated in this study are of comparable affinities to the
reported molecules (Figure 6d).
In the recent past, there have been notable developments in
cDNA display technology in terms of time required for the
execution of each round of cDNA display/selection, improvements in ligation reactions, restriction digestion, and increased
purified fusions for higher library size.31,37−41 The progress in
the faster execution of ligation reactions from 1 h to 10 min to
30 s is highly notable; however, the use of ribonucleases and
endonucleases should be avoided to refrain from any sort of
contamination that may negatively affect the whole selection
process.21,31,39 These methods require 2−3 days/round. In a
previous report, the purification steps have been modified to
increase the efficiency of the system by reducing the time of
processing to 2 days/round.38 Trap display smartly encounters
the process to tremendously reduce the execution time to 2.5
h/round;40,41 however, when using libraries, inclusion of
purification steps for obtaining full-length cDNA fusions is
important to avoid selection of prematurely terminated fusions
that can be a potential source of noise. Alternatively, custom
designed libraries that do not contain stop codons should be
used to counter this problem; however, it is noteworthy that
custom designed libraries are expensive to prepare. Furthermore, the untranslated mRNA-linker present in the unpurified
mixtures (∼70−80%) will participate in the selection process
by the formation of aptamers and thus interfere with the
outcome of selection.11,12,20 Recently, cDNA display was also
improved by the introduction of purification protocols that
increased the fusions by approximately 10-fold that can yield
libraries by a higher order.37 However, this method requires an
execution time of 2−3 days/round.
Next, we explored the efficiency of linker-N with a totally
random library to obtain affinity molecules for the cytokine
VEGF. For comparison with an earlier report,20 we chose to use
the 3F library (Figure 6a). We prepared a 3F library containing
3 × 1012 molecules and performed seven rounds of selection
against immobilized VEGF. Stringency of selection conditions
was applied in terms of reducing concentration of VEGF (1 μM
to 1 nM) and increasing the number of washes. After selection,
the library was analyzed by binding assays and sequencing of
clones.
In the binding assay analyzed by PCR, the R7 library protein
fusions showed significant binding to VEGF (more than 2%;
Figure 6b (2)) compared to a negative control that did not
contain VEGF (less than 0.2%; Figure 6b (1)). There is a
possibility of enrichment of the library via the nucleic acid
aptamers, i.e., mRNA/cDNA hybrids. We prepared an R7
mRNA/cDNA hybrid by reverse transcription of the mRNAlinker and tested for binding potential. There was no binding
observed for VEGF via the mRNA/cDNA hybrid (less than
0.2%; Figure 6b (3)). The R7 protein fusions also did not show
affinity for Fc (less than 0.2%; Figure 6b (4)), indicating target
(VEGF)-specific enrichment of the 3F library. In the binding
assay analyzed by ELISA, the R7 protein fusions showed 5-fold
higher signal for immobilized VEGF compared with the
negative control that did not contain VEGF and Fc as a
nonspecific protein (Figure 6c). This result also indicates that
the library was enriched specific to VEGF.
Analysis of the sequences of clones revealed that the
candidates were of three major groups: G-1 at 60% and G-2
and G-3 at 15% each. The affinities of these candidates were
measured by competitive ELISA method20,24 and found to be
in the range of 2.1−35 nM (Figure 6d).
The potential advantages of the cDNA display with linker-N
over “puro-linker” are summarized in Table 3. The major
advantage lies in the preparation of the library where 6 × 1012
molecules can be obtained per milliliter of lysate used with the
3F library compared with 4 × 1010 molecules20 owing to the
higher protein fusion yields. This library was prepared by NNS
codon (NA, T, C or G; SC or G) coding for random
amino acids that also contained stop codons. We prepared 3 ×
1012 per 0.5 mL in this study compared to 1.2 × 1011 per 3 mL
used in the earlier paper.20 The most interesting difference was
evident in the number of rounds of selection required that led
to similar sequence convergence (60−70%), which is seven
rounds in the case of linker-N compared with ten rounds in the
case of “puro-linker” (linker-N is approximately 25−30% more
efficient). Furthermore, it may be noted that by using same
target (anti-Flag M2 antibody), similar higher efficiencies (4fold) were established by this system over the previous cDNA
display method (Figure 5c). This may be attributable to the
reduction in “noise” (unwanted and unconverted cDNA/
mRNA hybrids) in the case of linker-N owing to the formation
of higher protein fusions. The time required for the whole
selection process with linker-N is 7 days, whereas it takes
around 30 days with other linkers, and this appears as a huge
advantage in light of the need to accelerate the discovery
■
CONCLUSIONS
We have developed a high performance and robust platform
based on cDNA display reinforced by a new modified linker-N
that enables rapid and highly efficient preparation of templates
and protein fusions for utilization in directed evolution. The
whole cycle can be accomplished in 6−8 h. We expect it to be
beneficial for iterative cycles for ligand discovery and evolution.
Ten rounds of selection can now be estimated in 2 weeks,
which can be conservatively supposed to outpace even phage
display-based methods (2−3 weeks) in addition to the cell-free
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for 1 h at 37 °C to generate capped transcripts, purified by
RNeasy purification kit (Qiagen), and quantified by absorbance
at 260 nm.
Prior to ligation, mRNAs were annealed to linker-N (ratio
1:1; 50 pmoles) by heating at 94 °C and gradient-cooling to 4
°C. Ligation was performed by the addition of 3 U T4 Kinase
(NEB, USA) and 20 U of T4 RNA ligase (Takara, Japan) at 25
°C for 10, 20, and 40 min. Ligation was confirmed by digestion
of the mRNA-linker conjugates with RNase H (Ambion,
Austin, TX, USA) that cleaves RNA hybrids. Ligation efficiency
was checked by denaturing polyacrylamide gel electrophoresis
(6% gel and 8 M urea) using FITC and Sybr Gold (Molecular
Probes, USA) staining on a fluoroimager (Bio-Rad, Hercules,
CA, USA). Ligation efficiency was calculated by the equation %
ligation = (A/A + B)×100, where A is the band intensity of the
ligated product and B is the intensity of the remaining mRNA.
cDNA Display. The mRNA-linker conjugates (100 nM)
were translated in a Rabbit Reticulocyte Lysate IVT Kit
(Ambion, Austin, TX, USA) (hereby abbreviated as RRL) and
Wheat Germ Extract (Promega, Madison, WI, USA) (hereby
abbreviated as WGE).
In the case of RRL, translation was performed for 10 min at
30 °C, and protein fusions (i.e., covalent linking of protein to
the puromycin moiety) were matured in the presence of 65
mM MgCl2 and 750 mM KCl at 37 °C for 2 h.13,20
In WGE, translation was performed for 10 min at 25 °C, and
proteins fusions were matured by the addition of 65 mM MgCl2
and 750 mM KCl at 25 °C for 1 h. Sampling was done for
electrophoretic analysis at 10 min (i.e., before addition of high
salts), 30 min, and 1 h (after addition of high salts). For
monitoring the effect of high salts, proteins fusions were also
matured in the absence of high salts for 1 h.
Before reverse transcription (RT), the protein fusions were
purified by immobilization on Ni-NTA magnetic beads
(Qiagen) for 10 min at 25 °C. The beads were washed with
1× PBS buffer, and RT was performed by the addition of 20 U
of M-MLV reverse transcriptase (Takara, Japan) at 42 °C for 10
min and placed on ice. After several washings, fusions were
eluted twice with 250 mM imidazole for 5 min at 25 °C. For
electrophoresis confirmation, the samples were incubated with
RNaseH (Ambion, Austin, TX, USA) at 37 °C for 30 min to
remove the mRNA. Analyses of all the samples were performed
by 12% SDS-PAGE containing 8 M urea and detected by FITC
on a fluoroimager. Efficiency of fusion formation was calculated
by the equation % fusion formation = (A/A + B)×100, where A
is the band intensity of the protein fusion (mRNA-linkerprotein) and B is the intensity of the remaining mRNA-linker.
Protein Labeling and Immobilization. Immunoglobulin
G (IgG) was purchased from Sigma, interleukin-6 receptor (IL6R) and VEGF were from PeproTech (London, UK),
acetylcholine binding protein (AChBP) was cloned from
Aplysia kurodai [manuscript in preparation], and the recombinant protein was expressed in E. coli and purified. These
proteins were biotinylated using EZ-Link Sulfo-NHS-SS-biotin
(Pierce, Rockford, USA) at a molar ratio of 1:10. Unreacted
biotin was inactivated with 1 M Tris-HCl (pH 8.0) and
removed by dialysis for 16 h at 4 °C. Biotinylation of proteins
was confirmed by SDS-PAGE and Western blotting using
streptavidin-HRP (GE Healthcare). Quantitative immobilization was checked by titration of biotinylated proteins against a
fixed amount of streptavidin-coated magnetic beads (Takara,
Japan) for 30 min at 25 °C. The supernatant was stored, and
the immobilized proteins were eluted with 100 mM
advantages. We consider this as a good starting point to achieve
versatility if combined with high-throughput automated systems
for rapid and exponential discovery of ligands and lead
molecules. For laboratory-scale throughput, as demonstrated
here, multiple samples can be processed in parallel using
microtubes/microplates far less laboriously and cost-effectively.
Potential applications can be in proteome exploration for
mapping multiple biomolecular interactions and in drug
discovery, an area that always requires better throughput
technologies for accelerated discovery and optimization of
biomolecules. Thus, we expect this cDNA display method to
provide higher diversity libraries for searching novel ligands at
reduced cost, time, and labor and to combine the intrinsic
throughput with high-throughput systems to accelerate the
discovery process. The recent developments, including the
optimized linkers and methods,31,37−41 and higher-throughput
systems42,43 are expected to provide necessary support and
significantly impact discovery research.
■
EXPERIMENTAL SECTION
Synthesis of Puromycin Linker. The puromycin linkeroligonucleotide (hereby designated as linker-N) installed with
multifunctional groups, 5′-CCCCCCCGCCGCCCCCCG(5Me-dC)A18(Spec18) (Spec18) (Spec18)(F-dT) (Spec18)CC(Puro)-3′, was custom synthesized (BEX, Tokyo, Japan). The
symbol 5-Me-dC denotes 5′-dimethoxytrityl-N4-(O-levulinyl-6oxyhexyl)-5-methyl-2′-deoxycytidine; Spec18, C18 spacer
phosphoramidite; F-dT, fluorescein-dT; and Puro, puromycin
CPG. Linker-N was synthesized by the DNA synthesizer
ABI394 (Applied Biosystems, Japan), and the 5′-end of the
linker was protected by acetylation. After synthesis, the levulinyl
protecting group in the 5-Me-dC residue was removed by 0.5
M hydrazine hydrate in 1:1 pyridine/acetic acid, and the
column was further washed by pyridine/acetic acid (1:1) and
then by acetonitrile. The primer sequence 5′-CCTG-3′ was
branched from the activated brancher 5-Me-dC in the linkeroligonucleotide. The product, i.e., primer-branched linkeroligonucleotide, was cleaved off from the column by K2CO3 in
methanol followed by deprotection of the acetyl group.
Additional deprotection was performed by reaction in 25%
ammonium hydroxide for 1.5 h at 65 °C. These conditions
were similar or milder than commonly used.44,45 The linkeroligonucleotide was purified by reversed-phase HPLC and
confirmed by TOF-MS and gel electrophoresis. All phosphoramidite reagents used for modifications were from Glen
Research (Sterling, VA, USA).
Oligonucleotides, Vectors, and Template DNA Constructs. The B-domain of Protein A (BDA)22 was amplified
from the pEZZ 18 protein A gene fusion vector (GE
Healthcare) and Pou-domain of Oct-1 (PDO), according to
the previous report.23 Three-finger (3F) was cloned from the
South American coral snake Micrurus coralinus,20 and 6R14 was
obtained from the pBADThio/TOPO vector IL-6R10-14.20
Custom oligonucleotides containing SP6 promoter, cap site,
Xenopus β-globin untranslated sequence (UTR), and translation
initiation codon (ATG) were added at the 5′ end and spacer
(G3S)2, 6xHis, G3S, and Y-tag sequences were added at the 3′
end by PCR. Amplification was carried out by denaturation (94
°C) for 20 s, annealing (60 °C) for 15 s, and extension (72 °C)
for 30 s.
Transcription and Ligation of Linker-N. Purified DNAs
were transcribed by SP6 RNA polymerase in the RiboMAX
Large Scale Production System (Promega, Madison, WI, USA)
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dithiothreitol (DTT; Wako, Japan) for 10 min at 25 °C. The
supernatant and eluted fractions were analyzed by SDS-PAGE
to confirm the ratio of streptavidin-coated magnetic beads and
proteins required for complete immobilization.
In Vitro Selection. The mRNA-(linker-N) conjugates of a
nonbinder and a binder were mixed at a ratio of 20:1,
translated, and processed to cDNA display proteins. In the case
of disulfide-rich ligands, such as 3F and 6R14, translation and
maturation was carried out in the presence of protein disulfide
isomerase (PDI) (1:1 molar ratio to display protein assuming
that more than 70% fusion is formed with respect to input
template), 10 mM oxidized glutathione (GSSG), and 1 mM
reduced glutathione (GSH).24 DTT was excluded from all of
the buffers. The His-tag eluted samples (50 μL) were mixed
with 150 μL of selection buffer (SB; 1× PBS, 100 mM NaCl,
and 0.1% Tween-20) and incubated with respective immobilized targets at 25 °C for 30 min. The beads were washed 10
times with 200 μL of the same buffer and eluted with 100 mM
DTT and desalted (Microspin columns, GE Healthcare). The
“before selection” and the eluted (“after selection”) fractions
were amplified by PCR using 0.2 μM of the primers 5′ATTTAGGTGACACTATAGAATACAAGCTTGCT-3′ and
5′-TTTCCCCGCCGCCCCCCGTCCTGCTTCCGCCGTGATGAT-3′ for 25 cycles (denaturation (94 °C), 20 s; annealing
(60 °C), 15 s; and elongation (72 °C), 30 s). Quantitative
analysis was performed by denaturing gel electrophoresis (4.5%
gel and 8 M urea), Sybr Gold staining, and fluoroimaging.
Selection Using a Mixture of Flag-Tag and 8-Mer
Peptide Library. An 8-mer peptide library was prepared by
constructing the oligonucleotide fragment containing the
random region flanked by complementary “overlap sequence”
on the 3′ end and His-tag and Y-tag sequences on the 5′ end.
The random region was coded by NNS codon (NA, T, C or
G; SC or G). The other oligonucleotide fragment contained
SP6 promotor and cap site at the 5′ end followed by 5′-UTR,
Kozak, and ATG. The two fragments were joined by overlap
PCR for 15 cycles of denaturation (94 °C) for 20 s, annealing
(60 °C) for 15 s, and extension (72 °C) for 30 s. The Flag-tag
construct was prepared in a similar manner except that the
random region was replaced by Flag-tag sequence. Sequences
were confirmed by cloning and sequencing.
For selection experiments, 10 pmoles of 8-mer library
mRNA-linker was mixed with 10 fmoles of Flag-tag mRNAlinker. Translation and maturation was performed in Wheat
germ extract (WGE) and processed to cDNA protein fusions.
For “puro-linker” the earlier protocol was followed.13 The
cDNA protein fusions were mixed with 200 nM of anti-Flag M2
antibody (Sigma, USA) in 200 μL of SB and incubated for 30
min at 25 °C. The beads were washed 5 times (Round 1), 8
times (Round 2), and 10 times (Round 3) with an equal
volume of SB and eluted with 100 mM DTT followed by
desalting. The eluted products were PCR amplified and
proceeded to the next round of selection for a total of 3
cycles. After Round 3, the PCR products were cloned (TA
cloning, Invitrogen) and sequenced by random picking of the
clones.
Selection of 3F Library against VEGF. A 3F library was
prepared according to the previous report.20 The library
mRNA-(linker-N) (200 nM) was translated in 0.5 mL of lysate
and processed to cDNA fusions. Estimation of cDNA fusions
was performed by SDS-PAGE with a known amount of mRNAlinker as a concentration standard. An estimated 3 × 1012
molecules were obtained as cDNA fusions.
The cDNA fusions were incubated with immobilized VEGF
for 1 h at 25 °C in 200 μL of SB followed by several washings
with SB and WB (1× PBS, 100 mM NaCl, and 0.5% Tween20). The bound molecules were eluted with 100 mM DTT.
The eluted fractions were desalted and PCR amplified
(denaturation (94 °C) for 20 s, annealing (60 °C) for 15 s,
and extension (72 °C) for 30 s). For applying selection
pressure, the concentration of VEGF was varied from 1 μM to
1 nM, and the number of washes was progressively increased
with WB. After Round 7 (R7), PCR products were cloned and
sequenced.
Binding Assays. R7 mRNA-(linker-N) (200 nM) was
translated and processed to cDNA fusions. These fusions were
divided into three parts and incubated with streptavidin beads,
VEGF beads (200 nM), and Fc beads (200 nM) for 30 min at
25 °C in 200 μL of SB. In the fourth reaction, mRNA-(linkerN) was reverse transcribed, and an equivalent amount was
incubated with VEGF beads. The beads were washed 10 times
with the same buffer and eluted with 100 mM DTT. The eluted
fractions were desalted and PCR amplified with the following
conditions: denaturation (94 °C) for 20 s, annealing (60 °C)
for 15 s, and extension (72 °C) for 30 s.
The R7 fusion proteins were also analyzed by the ELISA
method.24 R7 cDNA fusions were divided into 3 parts and
incubated with SA beads, 200 nM VEGF beads, and 200 nM Fc
beads in PBS-BSA (0.01% BSA) at 25 °C for 1 h. The mixtures
were washed with PBS-T (0.1% Tween) and subsequently
incubated with anti-His-antibody (R & D Systems) in PBS-T
for 30 min at 25 °C. The mixtures were then washed several
times with PBS-T followed by addition of the substrate,
3,3′,5,5′-tetramethylbenzidine (TMB; Sigma). After color
development, the reaction was stopped by the addition of 0.5
M H2SO4; the samples were centrifuged, and absorbance was
measured at 450 nm.
Measurement of Dissociation Constant. Dissociation
constants were measured according to the previously reported
method.24 A constant amount of 3F display protein (∼1 nM)
was incubated with varying amounts of VEGF (0.1 nM to 1
μM) for 1 h. The mixture was applied to a constant amount of
immobilized VEGF (∼100 nM) and incubated further for 30
min. After several washings, anti-His-antibody (R & D
Systems) was added at 1/2000 dilution and incubated for 1
h. The substrate TMB was added after several washings of the
incubation mixture. After proper color development, the
reaction was stopped by the addition of 0.5 M H2SO4, and
absorbance was measured at 450 nm.
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscombsci.5b00139.
Sequences of Round 0 (initial library) and Round 3
selection for anti-Flag M2 antibody (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Tel: +81 48 2621247. Fax: +81 48 2621248. E-mail:
[email protected].
*E-mail: [email protected].
Notes
The authors declare no competing financial interest.
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■
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ACKNOWLEDGMENTS
We thank S. Kobayashi for technical support. We gratefully
acknowledge Dr. Shozeb Haider, Queen’s University, UK, for
critical reading of the manuscript. This work was supported by
New Energy and Industrial Technology Development Organization (NEDO) (04A02542a), Japan to T.K. and in part by
Janusys Corporation to M.N.
■
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