For more information

Rebuilt Cell-Free System (PUREfrex®);
From basic research to industrial application
Takashi Ebihara, Ph.D.
COO
GeneFrontier Corporation

SEED 2023
30 May - 2 June 2023

For more information

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!

<Ex, Non-natural AA introduction>✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

Ready-made kit is coming soon!

2For more information

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!

<Ex, Membranewith Nanodisc; artificial membrane-like structure>
Non-naturalprotein
AA introduction>✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

Ready-made kit is coming soon!

3For more information

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!

<Ex, Membranewith Nanodisc; artificial membrane-like structure>
Non-naturalprotein
AA introduction>✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

Ready-made kit is coming soon!

4For more information

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!
✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

PURE system simulator
Matsuura T. et al. PNAS (2017) 114 (8) E1336
https://doi.org/10.1073/pnas.1615351114

5

For more information

Totally constructive, molecular based system-Customize expression toolbox for your research-

Only necessary molecules
for transcription/translation

Open theBlack box!

<Ex, Membranewith Nanodisc; artificial membrane-like structure>
Non-naturalprotein
AA introduction>✓ Rebuilt cell-free system✓ Tunable for your biologics✓ Simple, Fast
✓ Suited for High throughput system✓ Very low contaminants

Ready-made kit is coming soon!

6

-Having good productivity10-fold• Reaction at 37ºC for 4 h• 0.5 µL of reaction mix/ lane
• stained with Oriole (Bio-Rad) andanalyzed with an image analyzer (LAS)✓ Good expression for many proteins, small to large.
✓ Good purity with simple purification.
✓ Good productivity, ~g/L.
7

-KSF; AT rich codon on N-term-

Design of DNA template is important.
Manual is Free to download from our Web site here.
8For more information-Template, Temperature, Timever. 2 (4 mM GSH), 1 ng/µL DNA
reaction at 16/23/30/37ºC for 4/8/24 hours

sfGFP

M S K G E E L F T G
ATGAGTAAAGGAGAAGAACTTTTCACTGGA

sfGFP (ST) (NM3)M S K S T E L F T G
ATGAGTAAATCTACTGAACTTTTCACTGGA

9For more information-Improve process from Days to HoursPUREfrex® Synthesis

orUse forapplication
Mix solution
I, II and III

Addition oftemplate DNA

Incubation for 2-4 hours(Purification, if necessary)

Total: 2-4 hours

Synthesized DHFR
mg/mL

100mL package will be launched soon!

E. coli Expression
Transformation andovernight cultivation

Induction andexpression

Total: 3-4 days

Purification

Use forapplication

mL ～ L scale
Ver. 1.0

Ver. 2.0/2.1

Great Flexibility
from basic research
to industrial applications 10

For more information-Expression of scFv and FabSDS-PAGE

Activity+/For correct folding
Murakami et al. (2019) Sci. Rep. vol.9, p.671. (Supplementary Information)✓ Active scFv and Fab (derived from Herceptin) can be synthesized.
1

For more information-For HTS of mAb-

✓ Active Fabs are expressed/screened in HT manner.
12

For more information-For HTS of mAbiBody’s Ecobody Technology

Collecting B cells fromblood and tumor tissue
Active FabProtein

Selection of desired
single B cellsEcobody is…
・Human/Rabbit derived
・mAb in “Only 2” days!
・Fully in vitro screening
・No culture

Single-cell RT-PCR
amplification of clonal
antibody genes

Cell-freeExpression

Drug Discovery
Diagnosis
Reagent
Antibody
Evaluation

iBody’s Technology with two patents
1. High Expression with SKIK-tag
2. High Binding Activity with LZ-tag

Ojima-Kato et al. (2017) Sci. Rep., 7, 13979.
https://www.ibody.co.jp/en/✓ Active Fab is expressed/screened in HT manner.
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For more information-For display technologySDNC system is…
・for VHH/cyclic peptide
・with High diversity
・Fully in vitro screening

Evaluation
・ELISA
・SPR(BIAcore)・BLI(Octet)
・Cell based assay
https://www.epsilon-mol.co.jp/eng/✓ PUREfrex is applied for cDNA display based screening.
14

For more information-Application for BiTEanti-CD3-scFv/CD19-scFv

VL

VH

VH
VL

✓ 0.6mg/mL active BiTE was produced under optimized condition.
15

-Application for IgGWith a littlecustomization

Only 2 days for IgG!!
Murakami et al. (2019) Sci. Rep. vol.9, p.671.
Poster_PSSJ 2017

✓ Full size IgG can be synthesized.
16-Application for Cell based assay+/+ IL-2 DNA

As disulfide bridge enhancer

Synthesis of IL-2 protein

KHYG-1 cells+ synthesized IL-2
Cell cultivation with IL-2
Cell viability assay (WST assay)

Human IL-2
MW: 15.5 kDa
1 S-S bond

✓ Directly applicable to cell-based assay.
17

For more information-Application for complex molecule-Jensen et al. (2021) Protein Expr. Purif., 186, 105910.

✓ Functional insulin analogues can be synthesized.
18

-Application for complex molecule-Luciferase - Gaussia princeps (uniprot.org)

Poster_PSSJ 201819

For more information-Application for glycosylation; CFPS-IVGCell-freeProtein
Synthesis(CFPS)

(kDa)
15
10in vitro Glycosylation (IVG)

GlycosylatedProtein

UDP-/ Enzyme A

UDP-/ Enzyme B

CMP-/ Enzyme C-+-++-+++

Applicable to glycosylation of target proteins.
Various reaction system can be designed.

Poster_MBSJ 2021
20

For more information-Application for glycosylation; CFPS-IVG-

Solubilized membrane protein, hADRB2, are also
glycosylated by multi-step IVG

Poster_MBSJ 2021
21

For more information-Application for acetylationAcetylation (by NatB)

target: α-synuclein (K6A)
MDVFMAGLSK・・・

Target Protein
DNAAcetyl-CoA

Hisx6

Chromatogram of MS1 intensity
none+NatB/Ace-CoA-Ace

NatBDNA

Δ10-CHO-THF+Ace

Ace-MX
X=D/E/N/Q

Perfect control of N-terminal acetylation of α-synuclein
22For more information-Application for myristoylationMyristoylation (by NMT)

Functional N-terminal myristoylation confirmed by localization
23

For more information

Ribosome display system using PUREfrex◆ Advanced screening system
for Biologics• mAb (scFv / Fab)
• VHH
• Cyclic peptide◆ High Selection Efficiency• Completely molecular based system• >1012 diversity

Licensed technology under JP4931135 etc.

24For more information-Broad applications, yet to come!-

Dwidar et al. (2019) J. Am. Chem. Soc. vol.141, p.11103.

Levy et al. (2021) ACS. Synth. Biol. vol.10, p.609.

Fujita et al. (2023) ACS Nano. vol.17(4), p.3358.

Heames et al. (2023) Nature Ecology&Evolution. vol.7, p.570.
25

For more information-Broad applications, yet to come!-

Sakatani Y, et al. (2019) PEDS, 32(11) p481,
https://doi.org/10.1093/protein/gzaa011

Zimmermann I. et al. (2018) eLife, 7, e34317.

Godino E, et al. (2023) ACS Synthetic Biology
https://doi.org/10.1021/acssynbio.3c00074

Tsuboyama et al. (2022) bioRxiv.
https://doi.org/10.1101/2022.12.06.519132

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Contact information

Let’s have a chat at our Booth!!

For reagent use for expression / screening of biologics

https://purefrex.genefrontier.com/

For screening service / collaboration / technology transfer
for generation of new biologics
Takashi Ebihara, Ph.D., COO, GeneFrontier

E-mail: ebihara@genefrontier.com