Simple and Effective Selection of cyclic peptides by®
Ribosome display with PUREfrex
Kanehisa Kojoh, Kumiko Tsuihiji
GeneFrontier Corporation, Chiba, Japan④Sequencing after 4th round selection

Ribosome Display (RD) has many advantages over existing in vitro selection
methods in some ways, so we developed RD utilizing PUREfrex, which is a fullyreconstituted cell-free expression system, and named it as PUREfrex RD. We
have already reported that the PUREfrex RD showed higher selection efficacy
than that using other reconstituted cell-free expression systems or using S30extract systems, approximately with 100-fold or 1000-fold, respectively. We also
showed that selection of a scaffold protein or even a Fab format without
converting to scFv could be applied on PUREfrex RD. This year, we report theresult from the selection of cyclic peptides using PUREfrex RD. Cyclic peptides
are the peptides having a disulfide bond between two cysteine residues. The
natural examples are such as endothelin and atrial natriuretic peptide, andthose cyclic peptides show preferable drug like property than linear peptides.
There are several methods reported for the development of cyclic peptides, but
those methods need many steps and techniques (non-natural amino acids,enzymes, chemicals, etc.), and in most cases, it needs extra work for thescreening.
Here we report very simple and effective approach for the development ofcyclic peptides based on PUREfrex RD. A constrained 10 mer random peptide
library FLAG-Cys-X10-Cys-Myc was screened against biotinylated Erk2 protein(MAP kinase family protein relating to cell signaling) as a target antigen. The
nascent polypeptide-ribosome-mRNA complex (RD complex) was formed using
the customized PUREfrex supplemented with oxidized glutathione and E. coli
disulfide isomerase. After ~4 round selections, mRNA was recovered withgreatly increased outputs. Following cloning and sequencing resulted in goodvariety of the binders with some enrichment. Then, we established screening
process following the expression of those peptides in periplasm of E. coli as afusion protein. These fusion proteins had the binding activities for antigen
under the oxidized condition only without DTT. This result indicates that thefusion proteins hold ring structure with disulfide bond, and that the formationof a disulfide bond is necessary for its binding activity.
As such, we show that PUREfrex RD works for the selection of functional cyclic
peptides simply and effectively, and it could be applied for the development of
peptide based drugs, such as cyclic peptides or more constrained peptides
such as Disulfide Rich Peptide (DRP).

(Enrichment)(Unique)⑦Affinity measurement of selected binders by BIACORE<Condition>
・Sample: Cyclic peptide- MBP-fusion protien (Binder A and C)
・Antigen: Biotinylated Erk2 protein (immobilized RU=162)
・Sensor chip : SA(Alignment)Group 1Group 2Unique

Ribosome
Translation factors(Some of enrichment were observed and many unique clones were identified fromthe sequencing (94 clones). In addition, alignment analysis showed that some of
the clones were categorized into two groups as in the above. As an overview,arginine and hydrophobic amino acids such as leucine or tryptophan appeared withhigh frequency.

LeaderMBP

FLAGCys

MW:51KDa(His)8②Simple selection with PUREfrex RD for cyclic peptide
Ribosome Display complex with cyclic peptide

Cyclicpeptide<Host>
・E.coli (BL21DE3, Periplasm)<Culture conditon>・Temperature : 30 ℃
・Induction: final 0.1 mM IPTG

ReducedTarget antigen : Herceptin (anti-HER2 antibody)

Tetramer

250
150
15075

<Supposed application of binder>・Epitope mapping
・Anti-idiotype binder (like anti idiotype antibody)・Mimotope vaccination

Dimer

50

MonomerCys(NNS)10

C

c-Myc

LinkerHerceptin: IgG1

15

: Culture volume
10 µL /well
2nd antibody : anti-FLAG HRP: Culture volume
10 µL /lane
2nd antibody : anti-FLAG HRP
Detection: ECL prime (GE)<Condition>
・Target antigen : Biotinylated Erk2 protein

Frequency

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

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

Control Antibody A: IgG1Expression by PUREfrex

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.

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.

M A B C D A B C D

Tetramer

250
150
150
75

Dimer
Monomer

37

Control Antibody B: IgG4

Summary

Reduced50③In vitro selection of binders for the target, Erk2

Sequencing

Sequence

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.

SecMWhen 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.

Positive 68.4%

Loading

Loading

NonreducedCys(Enrichment of binders)(1st screening ELISA)

OD450

FLAGmRNA

Loading: Reaction mix 1 µL /well
2nd antibody : anti-FLAG HRP⑨Application II; For epitope mapping/mimotope development, etc.

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

SecM<Expression system>
・Customized PUREfrex

As 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-α
αor 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.

<Expression reagent>・PUREfrex (customized PUREfrex): final 2 mM・DTT
・Oxidized Glutathione (GSSG) : final 3 mM
・Disulfide isomerase (DsbC) : final 16 µM

NCyclic peptide(Binder A)<Condition>
・Temperature : 37 ℃
・Time
: 4 hour

2520PUREfrex 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.

Anti-TNF-α
α FabPeptide c-Myc
Cys

37

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

3.66X10-8
2.93X10-8

Lc Hc<Construct>

M A B C D A B C DBest suited forRibosome display

5.41X10-4
4.43X10-4<Construct>⑤Simple expression of cyclic peptide as MBP-fusion proteinNonreduced)

1.48X104
1.51X104⑧Application I; For bispecifics

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

KD (M)

Binders were purified by Ni resin. Binding kinetics of binders to Erk2 weredetermined using surface plasmon resonance with BIACORE. Biotinylated Erk2 wasimmobilized on a SA sensor chip according to the standard method. At a flow rate 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.

OD450

DNA or mRNA

kd (1/s)

Binder A
Binder C

Expression by E.coli①Best reconstituted cell-free protein synthesis system forin vitro selection having the lowest level of RNase
contamination: PUREfrex

ka (1/Ms)

OD450

Abstract

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.

Loading

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.

25
2015Loading: Reaction mix
1 µL /lane
2nd antibody : anti-FLAG HRP
Detection: ECL prime (GE): Reaction mix
2 µL /well
2nd antibody : anti-FLAG HRP

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.

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

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.

Recovered mRNA(On-going)(PUREfrex)(E.coli)(Condition)
Expression : 5 µL (reaction volume) /well
ELISA input : 2 µL /well(Condition)
Expression : 100 µL (culture volume)/well
ELISA input : 1 µL (culture lysis) /well

・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.

(Positive: 95 %)The peptide library with variety of 3 X 10 11 was used. Recovered mRNA
increased along with the progress of selection round. Final recovery rate
against input mRNA after 4th round was about 2.0 %.

(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.

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