In vitro selection from designed protein scaffold librarywith Ribosome Display on PURE system
Kanehisa Kojoh[1];Shizue Katoh[1,2];Akira Miyakoshi[1];Takashi Kanamori[1,2];Yuki Hayami[1];Kumiko Tsuihiji[2];Mikiko Nakamura[1,2];Tomoe Fuse[2];Takuya Ueda[2][1]R&D group, GeneFrontier Corp. [2]The Department of Medical Genome Sciences, Graduate
School of Frontier Sceinces, The University of Tokyo
Abstract⑩Constitutive nuclear localization of endogenous Erk2 by theErk2 binder containing NLS⑤Screening ELISA after PURE RD selection①PURE system: Protein synthesis Using Recombinant Elements
systemPURE system is a novel reconstituted in vitro transcription and translation
system which consist of purified 36 enzymes and E coli ribosomes necessary
for transcription, translation and energy recycling. It also contains amino acids,
NTPs and E.coli tRNA so the target protein can be synthesized just by additionof the template DNA to the reaction mixture.

MBP

FLAG-Tag

RNF8 scaffold

His-Tag

Construct

EGFPRNF8Condition
・Expression format:
MBP-fused RNF8 scaffold
・Host:
BL21(DE3)
・2nd Antibody:anti-FLAG(M2)-HRP

Anti-Erk2Hoechst
antibody

MergedCell

Factor Xa

Thioredoxin-binder(negative control)
Nucleus

Erk2-binder No.33

Erk2-binder No.06

s/nRibosome Display (RD) on the Protein synthesis Using Recombinant Elements(PURE) system (PURE RD) was reported as a powerful method for in vitro
selection of specific binders such as antibodies or antibody fragments. ThePURE system shows very little activities of nucleases and other inhibitory factors.
Therefore, mRNA/protein/ribosome ternary complex (RD complex) in the PURE
system is highly stable and the mRNA recovery rate is greatly increased ascompared with the conventional cell extract-based RD. Our current researches
on development of the new PURE system (PUREfrexTM) indicate that PUREfexTMbased RD (PUREfrexTM RD)has high potential to be a new standard method toscreen the specific binders from the peptide and protein library .
Here we showed results of PURE RD applying to newly designed protein
scaffold library based on the substrate recognition domain of human ubiquitin
ligase E3 protein. After 4th round in vitro selection of the binders toErk2(extracellular signal-regulated kinase 2) from this library, several uniquebinders were obtained. These Erk2 binders showed specific binding activity to be
used in general applications in vitro. In addition to that, the Erk2 binders showed
intracellular binding activity for Erk2 in the cytoplasm and inhibited thephosphorylation activity of Erk2 for nuclear protein p90RSK significantly inHEK293T. Also, the Erk2 binder with a Nuclear Localization Signal (NLS) resulted
in the nuclear translocation of Erk2. These results indicate that the Erk2 binders
could work not only in vitro but also in the cells as an intrabody.

EGFP-RNF8-fusion protein

Endogenous Erk2

Erk2-binder No.44Results
・Hit:64/95 (67.4%)
・ Uniqueness: total 14clones
No.44 (80%), No.33 (10 %),single clones (10%)

Screening ELISA after 4th round selection

After transfection of RNF8 scaffold expression vector, the EGFP-Erk2 binders
with NLS (nuclear localization signal) translocated the endogenous Erk2
protein from cytosol to nucleus. This result indicates that RNF8 scaffold may beable to regulate the localization of intracellular target protein in a strategic way
by addition of NLS or NES (nuclear exporting signal) to RNF8 scaffold.

⑪Development of highly purified PURE system (PUREfrexTM)
which has been more optimized to Ribosome Display⑥Overexpression and Purification
Overexpression
・Expression format : MBP-fused RNF8 scaffold
・Host : BL21(DE3)

Improvements
1, Highly purified protein factors by addition of futher column work.
2, Removal of Lipopolysaccharide (LPS) by treatment of detergent.

Purification
・Purification column: Amylose resin
・Final yield : 240 mg/L culture⑫The comparison of mRNA recovery rate for ribosome
display between the PUREfrexTM and Old PURE system⑦Determination of affinity by Biacore experiment

Fig.A: Antigen (+)

Fig.B: Antigen (-)

Fig.C: Antigen (+)/Antigen (-)

single cycle kinetics

PUREfrex

PUREfrex

PUREfrex②Ribosome Display complex in the PURE system (PURE RD)
N
Erk2 binder No.33

kon (M-1s-1)

koff (s-1)

KD (M)

2.3××105

7.0××10-3

2.9××10-8

RibosomemRNA
Scaffold protein

CLinker

Advantages:1) Ribosome Display complex is highly stable in the PURE system because
activities of nucleases and other inhibitory factors are very low in thissystem.
2) The mRNA encoding a scaffold protein with translation arrest sequenceof SecM remains stably attached to ribosomes in the PURE system.

③In vitro selection of binders for Erk2 protein using PURE RD

Binding kinetics of Erk2 binder to Erk2 was determined using surface plasmon
resonance with Biacore Biosensors T100. Biotinylated Erk2 was immobilized
on a SA sensor chip according to standard method. At a flow rate of 30 μl/min,six concentrations from 500 nM to 30 nM were used to record sensorgram andkon, koff and KD were determined by evaluation software.

⑧Immunoprecipitation of Endogenous Erk2 by Erk2 binder(pull down assay)
RNF8 scaffoldConstruct

mRNA Library:((6× 1010)× 1000
In vitro translation
by PURE system

RT-RCR

PURE RD
Selection Cycle
Isolation of mRNAIn vitro SelectionResin

LysateIP

Erk2 Erk2 Erk2(-) Trx-01 -33 -06 -44

Erk2 Erk2 Erk2(-) Trx-01 -33 -06 -44

Transfection

Lysis

anti-Erk Poly AbIP
Western
Blotting

anti-V5 mono Ab(detection of binder)

After transfection of expression vectors with Erk2 binder gene, HEK293T cells
were incubated for 24 hours, and were lysed by detergent. The Erk2 binderErk2 protein complex was isolated by Co2+ affinity beads, and were examinedby w estern blotting using anti-Erk polyclonal antibody and anti-V5
monoclonal antibody. As a result the expressed Erk2 binders could
specifically bind to endogenous Erk2 protein in HEK293T cells. This result
indicates that RNF8 scaffold could properly work as intrabody in cell.

AntigenElution⑨Inhibition of Erk2 phosphorylation activity by Erk2 binders④RNF8 (E3 Ubiquitin Ligase) FHA Domain scaffold LibraryFeature
・MW:18KDa・Localization:
Nucleus and Cytoplasm
・Structure:ββsandwich
・Loop:4 loops (NNS)

RNF8 is a E3 ubiquitin ligase which plays a critical role in the early DNAdamage response via the ubiquitination of histone H2A and H2AX. The global
fold of the RNF8 FHA (Forkhead associated) domain in an 11-stranded βsandwich structure with the phosphopeptide-binding surface comprised of 4loops. We have randomized the nucleotide sequences encoding those 4 loopsto NNS ( red lines ). Construction of library was carried out by overlapextension PCR of synthetic DNA fragments.

PMA- ++ + +Binder

Erk2 Erk2- - Trx-01 -33 -06

Total-p90RSK
phospho-p90RSK(S380)
phospho-p90RSK(T359/S363)

Signalphospho-Erk

Erk1/2

Inhibition

phospho-p90RSK(T573)
Total-Erk2

Condition
・Initial library size:Trx binder 1013 moleclues+ Erk2 binder No.33 103 moleclues
・Antigen:Biotinylated Erk2 protein
・Resin :Streptavidine Magnetic Beads
・Selection cycles: 3 roundVector

In vitro transcription
DNA LibraryV5-tag His-Tag⑬Model experiment of ribosome display selection withPUREfrexTM and Old PURE system

T573PP

S380P

p90RSKC-Myc
P P

53.7 %(51/95)

ND(0/95)

3rd round

80.0 % 29.5 %(76/95) (28/95)

Screening ELISA after 2nd round selectionMore than 50 % of selected clones could bind antigen after second roundselection using PUREfrex-based RD (PUREfrexTM RD), but using Old PURE
system could not select Erk2 binders at second round selection.

Summary
1, We developed a new designed protein scaffold library based on the RNF8FHA domain, and a number of Erk2 binders were selected from this libraryusing ribosome display with PURE system (PURE RD). It is noteworthy that
selected RNF8 scaffold could work properly as intrabody in cell. The
current intrabodies are developed by an antibody-based technology, whichis emerging as novel therapeutic molecules for the treatment of Alzheimer’s,Parkinson’s and Huntington’s diseases. We believe that RNF8 scaffold also
have a potential of new therapeutic molecules for these diseases.

EIK1
PTranscription

GFP (Binder)

2nd round2, Ribosome Display (PUREfrexTM RD) using Highly purified PURE system(PUREfrexTM) had high selection efficiency than that using normal PURE
system (Old PURE system). As a result, we will be able to select a lot ofbinders to antigens with high diversity in a shorter time than ever.

Nucleus

S363

Results of screening ELISA
PUREfrex
Olds/nSecM

Erk2 binder No.33 was used as positive control in ribosome display model
selection with or without antigen (Erk2 protein). The amount of mRNA wasdetermined by real-time PCR. As shown in Fig.A, the recovered antigen
specific mRNA from PUREfrexTM was increased at 2.2-fold. Also, the recovered
non-specific mRNA as a background from PUREfrexTM was decreased to 1/5(Fig.B). As a result, the selection efficiency of ribosome display using
PUREfrexTM raised approximately 10-fold higher than that using Old PURE
system.

Transcription

After RNF scaffold expression vector was transfected in HEK293T. The
expressed EGFP-Erk2 binders obviously inhibited the phosphorylation activityof Erk2 for nuclear protein p90RSK in phorbol 12-myristate-13-acetate(PMA)induced HEK293T cell.

Contact
GeneFrontier Corporation
URL:www.genefrontier.comE-mail:kojoh@genefrontier.com
contactus@genefrontier.com