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
Ribosome Display (RD) on the Protein synthesis Using Recombinant Elements (PURE)
system (PURE RD) was reported as a powerful method for in vitro selection of specificbinders such as antibodies or antibody fragments. The PURE system is a novel cell-freeprotein synthesis system reconstituted from the purified components (translation factorsand enzymes) necessary for the translation in E.coli. The PURE 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 as compared with the conventional cell extract-based
RD. Our current researches on the PURE system indicate that PURE RD has high potential
to be a new standard method to screen the specific binders from the peptide and protein
library such as scaffold library.
Here we showed results of PURE RD applying to our own 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 to Erk2(extracellular signal-regulated
kinase 2) from this library, several binders were obtained. These Erk2 binders showed
specific binding activity to be used in general applications in vitro. In addition to that, theErk2 binders showed intracellular binding activity for Erk2 in the cytoplasm and inhibitedthe phosphorylation activity of Erk2 for nuclear protein p90RSK significantly in HEK293T.
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 invitro but also in the cells as an intrabody.

PURE 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 sothe target protein can be synthesized just by addition of the template DNA to the reaction
mixture.

Development of “highly purified PURE system” which has
been more optimized to Ribosome Display

Screening ELISA after 4th round selection
MBP

FLAG-Tag

RNF8 scaffold

His-Tag

Improvements

Construct

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

Factor Xa

The comparison of the amount of contaminants in New andOld PURE system

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

Beta-galactosidase

RNases/n

Abstract

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

Screening ELISA after 4th round selection
LPS

Determination of affinity by Biacore experiment
single cycle kinetics
LPS,RNase and beta-galactosidase in thereaction mixture was examined. The
amount of all contaminants in the NewPURE system was significantly less thanthat in the Old PURE system.

konErk2 binder No.33(M-1s-1)

KD (M)

7.0×10-3

2.9×10-8

koff

2.3×105

The comparison of activity for protein-synthesis between theNew and Old PURE system(s-1)

Erk2 binder (No.33) was purified by amylose resin. Final yield of the purified binder(fused MBP) was 200-250 mg from 1L of culture. 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 torecord sensorgram and kon, koff and KD were determined by evaluation software.

Immunoprecipitation of Endogenous Erk2 by Erk2 binder(pull down assay)N

RNF8 scaffold

Construct

VectorRibosome
SecMmRNA
Scaffold proteinCV5-tag His-Tag

LysateIP

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

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

Transfection

Fig.A: Antigen (+)

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

RNF8 (E3 Ubiquitin Ligase) FHA Domain scaffold LibraryWesternBlotting

After transfection of expression vectors with Erk2 binder gene, HEK293T cells wereincubated for 24 hours, and were lysed by detergent. The Erk2 binder-Erk2 protein
complex was isolated by Co2+ affinity beads, and were examined by western blotting
using anti-Erk polyclonal antibody and anti-V5 monoclonal antibody. As a result theexpressed Erk2 binders could specifically bind to endogenous Erk2 protein inHEK293T cells. This result indicates that RNF8 scaffold could properly work as
intrabody in cell.

Inhibition of Erk2 phosphorylation activity by Erk2 binders
・MW:18KDa
・Localization:

PMA- ++ + +Binder

Erk2 Erk2- - Trx-01 -33 -06

Total-p90RSK

Nucleus and Cytoplasm

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

・Structure:βsandwich
・Loop:4 loops (NNS)

Condition

In vitro selection of binders for Erk2 protein using PURE RD

PC-MycP P

EIK1Pp90RSK

phospho-Erk

Transcription

GFP (Binder)

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

EGFPRNF8

In vitro translation
by PURE system

PURE RD
Selection Cycle

Anti-Erk2
Hoechstantibody

Thioredoxin-binder(negative control)
Nucleus

In vitro SelectionErk2-binder No.06

EGFP-RNF8-fusion protein

ResinCondition
・Initial library size:(1×1010)×1000 moleclues
・Antigen:Biotinylated Erk2 protein
・Resin :Streptavidine Magnetic Beads
・Selection cycles:4 round53.7 %(51/95)

Erk2-binder No.44

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 round selection
using New PURE system-based RD, but using Old PURE system could not select Erk2binders at second round selection.

MergedIsolation of mRNAElution

2nd round

SummaryCell

Erk2-binder No.33

AntigenResults of screening ELISA
NewOldTranscription

mRNA Library

RT-RCRP

S380

Constitutive nuclear localization of endogenous Erk2 by theErk2 binder containing NLS

In vitro transcription
DNA Library

Nucleus

S363
P・Initial library size:Trx binder 1013 moleclues + Erk2 binder No.33 103 moleclues
・Antigen:Biotinylated Erk2 protein
・Resin :Streptavidine Magnetic Beads
・Selection cycles: 3 roundErk1/2

Inhibition

T573

Total-Erk2

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

Model experiment of PURE RD selection with New and Old
PURE system

Signalphospho-p90RSK(T573)

RNF8 is a E3 ubiquitin ligase which plays a critical role in the early DNA-damage
response via the ubiquitination of histone H2A and H2AX. The global fold of the RNF8FHA (Forkhead associated) domain in an 11-stranded βsandwich structure with thephosphopeptide-binding surface comprised of 4 loops. We have randomized thenucleotide sequences encoding those 4 loops to NNS ( red lines ). Construction oflibrary was carried out by overlap-extension PCR of synthetic DNA fragments.

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

IPanti-V5 mono Ab(detection of binder)

FeatureFig.B: Antigen (-)

Lysisanti-Erk Poly AbLinker

The comparison of mRNA recovery rate for ribosome display
between the New and Old PURE systems/nRibosome Display complex in the PURE system (PURE RD)

The amount of synthesized DHFR in theNew PURE system increases more thanthat in the Old PURE system, especially,
with PCR products as a template DNA.

Endogenous Erk2

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

1, We developed a new designed protein scaffold library based on the RNF8 FHA
domain, and a number of Erk2 binders were selected from this library using 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 anantibody-based technology, which is emerging as novel therapeutic molecules for thetreatment 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.
2, Ribosome Display using Highly purified PURE system (New PURE system) had highselection efficiency than that using normal PURE system (Old PURE system). As aresult, we will be able to select a lot of binders to antigens with high diversity in ashorter time than ever.

Contact
GeneFrontier Corporation
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E-mail:kojoh@genefrontier.com
info@genefrontier.com