Simple and Effective Selection of cyclic peptides by®
Ribosome display with PUREfrex
Kanehisa Kojoh, Kumiko Tsuihiji, Shizue Katoh, Takashi Umeda
GeneFrontier Corporation, Chiba, Japan①Best reconstituted cell-free protein synthesis system forin vitro selection having the lowest level of RNase
contamination: PUREfrex
DNA or mRNA

Translation factors

Shimizu Y et al., Nat Biotechnol (2001) vol 19, p751-755<Construct><Construct>

Leader・Fully reconstituted.
・No cells, No bias.
・Fast, Easy, Pure.
・High throughput.
・Lower contaminants.
Very low level of RNase,patented as JP4931135()

Best suited for
Ribosome display

Ribosome Display complex with cyclic peptide

Cyclicpeptide

Cys

MW:51KDa(His)8

Anti-TNF-α
α FabCyclic peptide(Binder A)<Host>
・E.coli (BL21DE3, Periplasm)<Expression system>
・Customized PUREfrex<Culture conditon>・Temperature : 30 ℃
・Induction: final 0.1 mM IPTG
Nonreduced

ReducedAs an application of cyclic peptide, we looked into the combination with antibody tomake it bispecific. Selected cyclic peptide binding to Erk2 was simply fused to Cterminal of a heavy chain of anti-TNF-α
α Fab, and the each binding activity against TNF-ααor Erk2 protein were checked up by ELISA. As a result, this fusion protein bound to theeach antigen under non-reducing condition as designed, while only the specificbinding activity to Erk2 disappeared under reducing condition.

M A B C D A B C D

Tetramer

250
15015075

Dimer

50

Monomer

37Loading: Reaction mix 1 µL /well
2nd antibody : anti-FLAG HRP<Condition>
・Temperature : 37 ℃・Time: 4 hour

⑨Application II; For GFP-fusion protein (Add in to GFP)<Construct>: Culture volume
10 µL /lane
2nd antibody : anti-FLAG HRPDetection: ECL prime (GE)Loading: Culture volume
10 µL /well
2nd antibody : anti-FLAG HRP

Some of the enriched clones in sequencing were expressed in periplasm of E.coli
in very simple construct (FLAG - Cys - 10aa - Cys - c-Myc - His8) , but they could
not be detected in western blotting and ELISA (not shown). Therefore, we tried toexpress it as cyclic peptide-MBP fusion protein as in the above. The data ofwestern blotting (left above) indicated that the expressed fusion proteins werepolymerized under oxidized condition in periplasm of E.coli. That polymerizations
seemed to occur through the SS-bond because polymerization disappeared under
reducing condition. In addition, these fusion proteins had the binding activities forthe target under the oxidized condition only without DTT (as shown in ELISA data,right above). This result indicates that the fusion proteins hold ring structure withdisulfide bond, and that the formation of a disulfide bond is necessary for its
binding activity. On the other hand, binder B and D showed very weak signal,
which suggest that those clones might be dropped in the screening with thisexpression & ELISA.

Expression by PUREfrex

Cyclic peptide(Binder A)<Host>
・E.coli ;BL21(DE3)

Loading: Lysate 1 µL /well
Excitation Wavelength : 485 nm
Emission Wavelength : 535 nM<Condition>
・Temperature : 30 ℃
・IPTG
: 0.1 mM (final)

Cyclic peptide was fused to C-terminus of GFP. The GFP fusion protein could
specifically bind to Erk without losing their fluorecence. This result proposes thepossibility to creat of GFPs that can bind to any desired targets.

⑩Application III; For epitope mapping/mimotope development, etc.
<Supposed application of binder>・Epitope mapping
・Anti-idiotype binder (like anti idiotype antibody)・Mimotope vaccination<Expression reagent>・PUREfrex (customized PUREfrex): final 2 mM
・DTT・Oxidized Glutathione (GSSG) : final 3 mM
・Disulfide isomerase (DsbC) : final 16 µM

NonreducedGFP

Target antigen : Herceptin (anti-HER2 antibody)(Enrichment of binders)(1st screening ELISA)
Herceptin: IgG1

<Reaction>
・Temperature: 37 ℃
・Time: 2 hour

Positive 68.4%

Sequencing

ReducedM A B C D A B C DmRNACc-Myc

Linker

SecMDimerMonomer

5037CysControl Antibody A: IgG1

Tetramer

250
150
150
75

<Condition>
・Target antigen : Biotinylated Erk2 protein

Loading: Reaction mix
1 µL /lane
2nd antibody : anti-FLAG HRP
Detection: ECL prime (GE)Loading: Reaction mix
2 µL /well
2nd antibody : anti-FLAG HRP

Recovered mRNA

(PUREfrex)(E.coli)(Condition)
Expression : 5 µL (reaction volume) /well
ELISA input : 2 µL /well

The peptide library with variety of 3 X 10 11 was used. Recovered mRNA

④Sequencing after 4th round selection(Unique)(Condition)
Expression : 100 µL (culture volume)/well
ELISA input : 1 µL (culture lysis) /well(Positive: 95 %)(Positive: 26 %)Due to the good expression property with PUREfrex, it showed remarkably better
hit rate than that with E. coli. The data suggests that PUREfrex is more suitable forthe expression and the screening at the initial process, not to miss the potentialbinders.

(Alignment)Group 1Group 2⑦Affinity measurement of selected binders by BIACORE

Unique<Condition>
・Sample: Cyclic peptide- MBP-fusion protien (Binder A and C)
・Antigen: Biotinylated Erk2 protein (immobilized RU=162)
・Sensor chip : SA

Binder A
Binder C

Some of enrichment were observed and many unique clones were identified
from the sequencing (94 clones). In addition, alignment analysis showed thatsome of the clones were categorized into two groups as in the above. As an
overview, arginine and hydrophobic amino acids such as leucine ortryptophan appeared with high frequency.

56/89
5/89
5/89
5/89
4/89
4/89
1/89

15

⑥Comparison of 1st screening ELISA between PUREfrex andE. coli expression

increased along with the progress of selection round. Final recovery rate
against input mRNA after 4th round was about 2.0 %.

Binder A
Binder B
Binder C
Binder D
Binder E
Binder F
Binder G

20

Cyclic peptide-MBP fusion proteins were expressed in PUREfrex. In this case, thepolymerizations were remarkably suppressed than that in E.coli expression (asshown in western blotting, left above), and all the binders showed good binding
activity (as shown in ELISA, right above). Those results suggest that PUREfrex issuitable for the expression of those binders.

③In vitro selection of binders for the target, Erk2

Frequency

Control Antibody B: IgG425

When the arrest sequence of SecM at 3''terminus is translated in RUREfrex, aribosome can be fixed strongly on mRNA. Also, release factors (RF1, RF2, RF3,RRF) were removed from PUREfrex, and oxidized glutathione (GSSG) anddisulfide isomerase from E.coli (DsbC) in optimized concentration to form
disulfide bond were added into PUREfrex. As a result, RD complex become
highly stable, and cyclized peptide is displayed on ribosome with highefficiency.

Sequence

OD450

SecM(Enrichment)

Lc Hc

Expression by E.coli

NCys

Peptide c-Myc
Cys

Loading②Simple selection with PUREfrex RD for cyclic peptide(NNS)10

FLAG

15

PUREfrex was developed as highly purified PURE system , and it is clearly
more suitable for Ribosome Display. PURE system is a w ell-know nreconstituted in vitro transcription and translation system which consists ofpurified 36 proteins and E. coli ribosomes necessary for transcription,translation and energy recycling. It also contains amino acids, NTPs and E. colitRNA, so the target protein can be synthesized just by addition of the template
DNA to the reaction mixture.

FLAGMBP25
20

PointsRibosome⑧Application I; For bispecifics (Fab + Cyclic peptide)

OD450

Cyclic peptides are the peptides internally linked with at least one covalentbond, such as disulfide bond that generate the ring structure. There are several
methods reported for the synthesis of cyclic peptide library, but those methods
need many steps and complicated techniques (introducing of non-naturalamino acids, using of enzymes or chemicals to cyclize peptides etc.), and in
most cases, it needs extra work for library screening.
Ribosome display (RD) has many advantages over existing in vitro selection
methods, so we developed RD utilizing PUREfrex, a fully reconstituted cell-freeexpression system, and named it as PUREfrexRD. Here we report very simple
and effective approach for the selection of cyclic peptide using PUREfrexRD.
Our approach is as follows: A constrained 10mer random peptide library wasconstructed and screened against a target antigen. The cyclic peptideribosome-mRNA complex was formed using the customized PUREfrex. After
four round selections, following cloning and sequencing resulted in a large
variety of the binders with some enrichment. Then, we established screening
process by the expression of those peptides in periplasm of E. coli as a fusion
protein. These fusion proteins had the specific binding affinities for antigen
only under the oxidized condition without DTT. This result indicates that thefusion proteins hold the ring structure with disulfide bond, and that theformation of a disulfide bond is necessary for its binding affinity.
As such, we showed that PUREfrexRD works for the screening of functional
cyclic peptides simply and effectively, and it could be applied for thedevelopment of peptide based drugs.

⑤Simple expression of cyclic peptide as MBP-fusion protein

OD450

Abstract

Cyclic peptides were selected against anti-Her2 monoclonal antibody. A lot ofbinders were obtained from 1st screening ELISA, and some of the enrichments wereobserved as shown above. The binders didn’t bind to control antibody A or B
classified into IgG1 or IgG4, respectively. This result shows that those binders bind
only to the CDR regions of Herceptin specifically, not to the constant or Fc region.

Summary
1, Simple and effective selection system for the development ofcyclic peptide were reported.
・PUREfrex (fully reconstituted, customized, the lowest RNasecontamination) is a key to success.
2, Actual example of the selection of cyclic peptide against Erk2
as target was performed.
・Many, various, unique binders were obtained in just 4 round panning.
・10-8 KD binders were selected without affinity maturation.
3, Simple screening is possible with PUREfrex.
・Only with natural amino acids, expression is easy.
・Many clones can be expressed for the screening in parallel.
・PUREfrex makes it more effective comparing to E. coli.
4, Unique applications were proposed.
・Cyclic peptide can add another binding activity to the existing
biologics to make it bispecific in very simple manner.
・Targeting existing antibodies, epitope mapping, mimotope
development, anti-idiotypic binder (like anti-idiotypic antibody)development can be done with PUREfrex RD.

(On-going)ka (1/Ms)kd (1/s)

KD (M)

1.48X104
1.51X104

5.41X10-4
4.43X10-4

3.66X10-8
2.93X10-8

Binders were purified by Ni resin. Binding kinetics of binders to Erk2 were
determined using surface plasmon resonance with BIACORE. Biotinylated Erk2
was immobilized on a SA sensor chip according to the standard method. At a flowrate of 20 µL/min, five concentrations from 0.3 µM to 4.2 µM were used to record
sensorgram, and ka, kd and KD were determined by evaluation software.

・Unique cyclic peptide for PD-1 (and for other therapeutictargets) are under validation.
・DRP (Disulfide Rich Peptide) screening is on-going aiming
for the development of orally available biologics.
<Ackowledments>
We thank Mr. Keita Iguchi in KANEKA corporation for BIACORE analysis.

Contact
URL:www.genefrontier.com
E-mail:contactus@genefrontier.com