In vitro production of Fab, Toxin, and Immunotoxin using
PUREfrex® 2.0 with increased productivity○Satoshi Murakami, Rena Matsumoto, Takashi Kanamori
GeneFrontier Corporation (Chiba, Japan)

Abstract

3. Application of PUREfrex® 2.0

For more information, please contact us.
URL: www.genefrontier.com
E-mail: purefrex@genefrontier.com

Cell growth inhibition assay; can be applicable for synthesis/screening for scFv,Fab, Protein toxin and Immunotoxin

PURE systemは大腸菌の転写・翻訳系の構成因子を個別に精製して再構 成した無細胞タンパク質合成系である。PURE systemはRNaseやプロテアーゼなどの混入がほとんど無い上、目的に応じて反応液の組成

150を最適化し、従来に比べてリポ多糖（LPS）混入量を1/1000に減少させた新規PURE system (PUREfrex 1.0)

Cell: SK-BR-3
HER2 level: +++Culture: 4 days

scFv and Fabを開発した。また、分子シャペロン等の添加により、単純タンパク質だけでなく、SS結合を有するタンパク質

Cell Viability (%)

を自由に変更できるなど、他の合成系にはない特徴を有している。これまでに我々は、構成因子の精製法や不溶化しやすいタンパク質を活性型として合成可能であることも示した 。さらに反応液組成の見直しなどにより、PUREfrexのタンパク質合成能力を飛躍的に向上させることに成功した（PUREfrex 2.0）。本発表では

100

00.001

PUREfrex 2.0の利用例として、抗HER2 抗体Fabや毒素タンパク質（Gelonin、PE38など）、およびそれらをリンReaction mix:+カーでつないだImmunotoxinを合成し、機能評価を行った。PUREfrex 2.0によるFabの合成量は約0.5 mg/mL+(1 μM DsbC, 3 mM GSSG)

IC5050Protein synthesis

0.010.1

110

Concentration of product [nM](5 μM DnaK, 1 μM DnaJ, 1 μM GrpE)
150

Template DNA:PCR product (final 10 nM) with C terminal FLAG and His-tag

ク質として合成でき、真核細胞由来の無細胞タンパク質合成系を用いたアッセイではタンパク質合成を阻害scFv (single template DNA)する活性を示した。これらの融合タンパク質であるImmunotoxinは、Fabと毒素タンパク質の両方の機能を示Fab (Mixed template DNA)LC DNA
2 nM++HC DNA
8 nM

svFv DNA

10 nM
した。さらに、合成産物をHER2高発現株のSK-BR-3に未精製の状態で添加したところ、Fabや毒素タンパク質単独では増殖抑制効果はなかったが、Immunotoxinでは添加量に応じた増殖抑制効果が確認された。こCell Viability (%)

で、大腸菌で生産したFabと同等の結合能力を示した（EC50=0.5～0.6 nM）。毒素タンパク質は可溶性タンパ

Cell: BT-474
HER2 level: +++Culture: 6 days

37°° C for 6h

Incubation:

れらの結果は、PUREfrex 2.0 を用いることでImmunotoxinのような複雑なタンパク質を簡便かつ機能評価に10000.001

十分な量で合成できることを示している。

Amino Acids

Translation Factors
tRNAs

TALON® Metal Affinity Resin (Clontech)
50 mM Tris-HCl (pH 8.0), 500 mM NaCl,
20 mM MgOAc, 5 mM Imidazole

Wash
Buffer1

Binding buffer + 5 mM ATP (Incubate at30°C for 10 min, 3 times)

Wash
buffer250 mM Tris-HCl (pH 8.0), 500 mM NaCl,
5 mM Imidazole (Incubate at 4°C for10 min, 3 times)

Elutionbuffer

50 mM Tris-HCl (pH 8.0), 500 mM NaCl,
50 mM Imidazole

[kDa]

[kDa]

150

150

100

100

75

75

50

50

37

37

NTPs

25

25

2020

VL

15

The PURE (Protein synthesis Using Recombinant Elements)
system is a reconstituted cell-free protein synthesis system,
which consists of only purified factors necessary fortranscription, translation and energy regeneration.

10-20010-20020-1,000ContaminationRNase

very HighLow

very Lowvery Low

Endotoxin (LPS)very HighHighvery Lowvery Low

OKOK1.9

Abs at 450 nm

100-1,000

Abs at 450 nm

Typical Yield (μg/mL)

VL

VH

CLCH10.9
0.4

0.01

0.1

110100FabEC50

1.4

10

anti-CD63

Merge + DAPI

37°° C 3H

37°° C 3H

anti-HER2-FabRecycling
endosome

HC-Gelonin/LC

Golgi

HC-PE38/LC

Early
endosome

0.90.4CD63
0.001

10000.01

Concentration of product [nM]Template DNA

37°° C 3H

0H-0.1-0.10.001

1

anti-human FabInternalization analysis

anti-HER2-scFv
anti-HER2-Fabanti-HER2-Fabproduced by E. coli

2.4EC50

1.40.1

Concentration of product [nM]HER2anti-HER2-scFv
1.90.01

HER2 positive breast cancer cell lines were cultured with the medium (DMEM, 10% FBS, Penicillin/Streptomycin)
× 106) at 37°C for 4-6 days.
containing PUREfrex 2.0 reaction products (without purification, 1:400 to 1:1.64×
Cell viability was determined by WST-8 method (Cell Counting Kit-8, Dojindo). Mean of triplicates is shown.

ELISA (2nd Ab: anti-human Fab-HRP)

anti-HER2-Fab

2.4NewIC5050HCLC

2.9

Reconstituted system
PURE system(original*)

10102.9

S30 system

110000.001

1510

ELISA (2nd Ab: anti-FLAG-HRP)

Extract system

VH

Cell: MCF-7
HER2 level: +Culture: 4 days

10-20% gradient SDS-PAGE gel, Oriole staining“no DNA” and “Synthesized product”: 0.5 μL of reaction/lane“Purified scFv”: 0.3 μg/lane, “Purified Fab”: 0.2 μg/lane

E. coli based cell-free protein synthesis systems

0.1Concentration of product [nM]

Cell Viability (%)

Resin
Bindingbuffer

DNA
Ribosome

0.01

150

Batch purification

1. PUREfrex®; based on the PURE system technology

IC50

anti-HER2-Fabanti-HER2-scFv

oranti-HER2-FabGelonin
HC-Gelonin/LC
PE38
HC-PE38/LC

50

0.1

1101001000

Lateendosome

Concentration of product [nM]ER

Plasmid DNA

OK

OK

PCR product

NG

OK

OK

OK

Customization ofReagent

Difficult

Easy

Easy

Easy

OKNG

OK

OK

Ribosome display△○◎mRNA display

△○

◎○
○

Purification ofHis-tagged productAntigen: Extracellular domain of human ErbB2/HER2 (1 ng/well)Cytosol

Lysosome

Template
DNA [nM]

ProteinMW [kDa]

Synthesized Protein

EC50[mg/mL]

[μM]

[nM]

1028.00.41

14.50.5LC223.5

826.0

0.5

8.4

0.5-0.6

HCIn vitro display

anti-HER2-scFv*Reference; Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751

anti-HER2-Fab

PUREfrex 2.0(no DNA)

Retrogradetransport

Summary (scFv and Fab)BT-474 cells were surface-labeled at 4°C for 60 min with the binding medium (DMEM, 3% BSA, 20 mM HEPES (pH 7.4)) containing
PUREfrex 2.0 reaction products (without purification, 1:400). Cells were washed five times with the binding medium and incubatedat 37°C for 3h. Cells were then fixed and processed for dual-label indirect immunofluorescence microscopy. CD63 (Lamp3) is marker oflate endosomes and lysosomes. White arrows indicate partial co-localization of Fab or Immunotoxin with CD63. Bar indicates 20 μm.

Summary (Protein toxin and Immunotoxin)

EC50: 50% effective concentration of target binding activity.

2. How to use PUREfrex®

Protein toxin and Immunotoxin; for “Easy” and “High throughput” preparation of ProteinsGelonin
LCHC-Gelonin/LC+

Template DNA:ORF
T7 terminator(5 μM DnaK, 1 μM DnaJ, 1 μM GrpE)

PCR product (final 10 nM)
Toxin (single template DNA)

T7 promoter

MetToxin DNA

or

10 nM

...TAATGAATAACTAATCCStop

PCR product containing T7 promoter
and SD sequence

Immunotoxin (Mixed template DNA)LC DNA
1 nM++
HC-Toxin DNA
9 nM

24.7
53.2
37.4

LC124.7

HC-PE38962.5

Gelonin*1; is a 30-kDa protein from plant Gelonium multiflorum.
; inactivates 28S rRNA of eukaryote ribosome via its
N-glycosidase activity.

Gelonin andImmunotoxin

PE38 andImmunotoxin

8-X μL7-X μL(Amino acids, NTPs,tRNAs, etc.)

10 μL

10 μL

Solution II(Translation factors)

1 μL

1 μL

Solution III(Ribosome)

1 μL

2 μL

Water

[kDa]

[kDa]

150

150

100

10075

Template DNA (1-3 ng/μL per 1 kb)

X μL

X μL

20 μL

20 μL

37LC2520; is a fusion protein, in this study, fused “protein toxin”
to “anti-HER2-Fab” via glycine-serine linker (G4S).
*Reference;
1: Stirpe F. et al. (1980) JBC. vol. 255, p. 6947
2: Kreitman RJ. (2009) BioDrugs. vol. 23, p. 1

CLCH1

201510VLVH

CLCH1Abs at 450 nm
Controlno DNA

PE38

HC-PE38/LC

Gelonin

○0.5

ND

ND

ND

4.7

○

ND

ND

ND

65.0

○

1.0

0.6

0.25

ND

・Development of a novel protein toxin.

1.9

Our Products

PE38
HC-PE38/LC

EC50

1.4
0.9/

0.4

HC-Gelonin/LC

2.7

Gelonin
HC-Gelonin/LC

anti-HER2-Fab

Fluorescence of synthesized GFP(relative value)

ND

・Improvement of binding affinity and cytotoxicity of Immunotoxins.

anti-HER2-Fab
2.4

GFP synthesized by using HeLa cell-extract based cell-freeprotein synthesis system (1-Step Human Coupled IVT KitDNA, Thermo scientific) with PUREfrex 2.0 reaction
products (without purification, 1:1000).

ND

・Synthesis of “full-length IgG”.

ELISA

1000ND

On-going Project

2.9

Control + PUREfrex 2.0 reaction products④ Analyze the synthesized product.
“Direct assay without purification”

VH

10Translation Inhibition assay

37°37°C

0.44

MCF-7ND・PUREfrex 2.0 reaction products (without purification) were applied“directly” to living cells for the cytotoxicity assay of immunotoxin.

LC25VL10-20% gradient SDS-PAGE gel, Oriole staining
Sample: 0.5 μL of reaction/lane

50

0.17

BT-474○・“Protein toxin” and “Immunotoxin” were synthesized, showing theactivity in vitro.

50

15

・If necessary, add “DS supplement”, “DnaK Mix”, and/or “GroE Mix”
to the reaction mixture.
・“Co-synthesis of multiple protein” is possible by using the mixed
template DNA.

③ Incubate the tube at 37°°C for >2 hours.

0.33

SK-BR-3PE3837Gelonin

Immunotoxin

8.0

IC50 [nM][nM]・LC and HC protein of anti-HER2-Fab were co-synthesized by using
the mixed template DNA.

75HC-Gelonin

50; is a 38-kDa truncated Pseudomonas exotoxin (PE)containing amino acids 253-364 and 381-613.
; inactivates eukaryote elongation factor 2 via its
ADP-ribosylating activity.

0.23

EC50

・His-tagged anti-HER2 “-scFv” and “-Fab” protein were synthesized in
the active form and purified using the affinity resin.

HC-PE38

PE38*2Translation
InhibitingactivityConclusion

T7 promoter-driven protein
expression vector for E. coli

② Mix the PUREfrex reaction mixture in a tube as follows.

[μM]

EC50: 50% effective concentration of target binding activity. IC50: 50% inhibitory concentration of cell growth. ND: Not detected.

37°° C for overnight

Incubation:

more than 10 any nucleotides

Solution I

1HC-PE38/LC

5’-GAAATTAATACGACTCACTATAGGGAGACCACAACGGT
TTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATSD sequence
ATACCAATG...
ORF

29.3

Synthesized Protein[mg/mL]+(1 μM DsbC, 3 mM GSSG)

T7 promoter

109PE38

Reaction mix:Protein
MW [kDa]

10HC-GeloninProtein synthesis① Prepare the template DNA.

Template
DNA [nM]

-0.1
0.001

Newa regular kit for the synthesis of proteins without disulfide bonds
0.01

0.1

1

10100

Concentration of product [nM]a supplement for the synthesis of proteins containing disulfide bonds
Antigen: Extracellular domain of human ErbB2/HER2 (1 ng/well)
2nd Ab: anti-human Fab-HRP 2nd antibody (Jackson ImmunoResearch)/a supplement for the synthesis of aggregate-prone proteins