Development of a method for the synthesis of aglycosylated full-length IgG using PUREfrex
再構成型無細胞タンパク質合成システムPUREfrexを用いた全長IgG抗体の合成法開発○Satoshi Murakami, Rena Matsumoto, Takashi Kanamori
GeneFrontier Corporation (Chiba, Japan)
Abstract

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URL: www.genefrontier.com
E-mail: purefrex@genefrontier.com

What’s PUREfrex® ?

Aglycosylated IgG (including IgG1, IgG2 and IgG4 subclasses) were synthesized using PUREfrex. The PURE (Protein synthesis Using RecombinantElements) system is a reconstituted cell-free protein synthesis system, which consists of only purified factors necessary for transcription, translationand energy regeneration. Recently, we developed an updated PURE system with higher productivity, which was launched as “PUREfrex 2.0” in 2015.
We reported that PUREfrex 2.0 could be used for production of fragment antibodies such as Fab and scFv by last year. Here, we report the further
application using PUREfrex 2.0 for production of IgG. We chose Trastuzumab as a model protein and optimized the composition of PUREfrex 2.0
reaction mixture and the reaction conditions as below; 1) adding disulfide bond isomerase (DsbC); 2) choosing GSH as an effective reductant; 3)adjusting GSH/GSSG ratio; 4) adding molecular chaperone (DnaK mix); 5) long-time incubation for 28h. We also suggested that synthesis temperatureand template DNA ratio (light chain/heavy chain) should be optimized for individual IgGs for the best yield. At the best mode of synthesis, theproductivity of Trastuzumab reached to 124 µg/mL. Moreover, the 68 µg of purified Trastuzumab was obtained from 1 mL of the reaction mixture after
the purification by protein A resin and the following gel filtration. The purified Trastuzumab exhibited high binding affinity to recombinant HER2
protein (KD=4.24E-10 M) and internalized into HER2 expressing BT-474 cells. Furthermore, other IgGs including IgG1, IgG2 and IgG4 subclasses werealso synthesized and confirmed their binding activity in ELISA. These results indicate that PUREfrex will be useful tool for high-throughput
production/screening of functional antibodies (scFv, Fab, and IgG).

PUREfrex® is a reconstituted cell-free protein synthesis system based on the PURE system* technology.
PUREfrex® consists of only purified factors necessary for transcription, translation and energy regeneration.
Advantage

Application

Low level of contamination
Easy adjustment of the reagent composition
PCR products usable as a template DNA

High-throughput preparation of proteins (including Fab, scFv, protein toxin etc.)
Protein science research
In vitro display (Ribosome display, mRNA display etc.)【Reference】 *: Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751

DNA
Ribosome Amino Acidsor

Our Products

Translation Factors
tRNAs NTPs

Coming soon//a regular kit for the synthesis of proteins without disulfide bonds

a supplement for the synthesis of proteins containing disulfide bonds/
IgG

1. Optimization of the synthesis of Trastuzumab using PUREfrex®

[kDa]

[kDa]

250

100

#6: Synthesis temperature

Synthesized protein [µg/mL]

[kDa]
250
150
100

Light chain

10
0
0

5

10

15

Heavy chain

ReducedSDS-PAGE

150

0

40
20
0
0

2

4

50

0

4

8

12

16

20

24

28

32

Light chain

Heavy chain

200
150
100
50

250

250
150

150

100

8

12

IgG

16

20

24

28

Heavy chain

IgG

40

*1: Goemans C. et al. (2014) BBA. vol. 1843, p. 1517

20

*2: Castanié-Cornet MP. et al. (2014) BBA. vol 1843,
p1442

0

FkpA

Skp

Incubation

Template DNA
ratio (LC:HC,
total 10 nM)

IgG Yield[µg/mL]PUREfrex 2.0 (normal version, 2 mM DTT)

37°°C for 16h

1:1

ND

DsbC concentration

PUREfrex 2.0 (normal version, 2 mM DTT)
with 3 mM GSSG, 0-20 µM DsbC and DnaKmix (x1)

37°C for 16h

1:1

35±
±2

3

Reducing agent

PUREfrex 2.0 (without DTT) with 2 mM
reducing agent, 3 mM GSSG, 5 µM DsbC andDnaK mix (x1)

37°C for 16h

1:1

55±
±1

4

GSH/GSSG ratio

PUREfrex 2.0 (without DTT) with 2 mM GSH,
0-12 mM GSSG, 5 µM DsbC and DnaK mix(x1)

37°C for 16h

1:1

74±
±12

5

Molecular chaperone andchaperone-like proteins

PUREfrex 2.0 (without DTT) with 2 mM GSH,
3 mM GSSG, 5 µM DsbC and chaperones

37°C for 16h

1:1

75±
±7

6

Synthesis temperature

Optimized reaction mixture (See No. 9)30, 37 or 42°°C
for 16h

1:1

70±
±11

7

Incubation time

Optimized reaction mixture (See No. 9)30 or 37°°C
for 0 - 32 h

1:1

74±
±6

8

Template DNA ratio

Optimized reaction mixture (See No. 9)

37°C for 28 h

1:1, 1:2, 1:4, 1:8

9

After optimization

PUREfrex 2.0 (without DTT) with 2 mM GSH,
3 mM GSSG, 5 µM DsbC and DnaK mix (x1)37°°C for 28 h

1:2 - 1:4

No

Composition of the reaction mixture

1

Before optimization

2

75

DnaK

37

20

124±
±9

Aglycosylated IgG (Trastuzumab) was synthesized using PUREfrex 2.0 under various conditions. After synthesis, all samples were centrifuged
at 9,100 xg for 10 min and 1.0 µL of supernatant was applied to non-reduced (10% gel) or reduced (12.5% gel) SDS-PAGE. The gels were
stained with Oriole fluorescent gel stain (Bio-Rad) and the protein bands were quantitated using LAS-4000 system (GE Healthcare). Yields are
expressed as means ± SD (n=3). ND, not detectable.

Synthesis temperature100(148.3 kDa)

75
50

50

Synthesized protein [µg/mL]

[kDa]

IgG
Heavy chain(50.6 kDa)

Template DNA ratio (Light chain/Heavy chain)

Cetuximab

Panitumumab

Nivolumab

Adalimumab

Cetuximab

Panitumumab

Nivolumab
LC:HC

37

Purification summary【Reference】

150

25

Adalimumab
Commercial
Trastuzumab

Purified IgG (Step 3)

Commercial
Trastuzumab

75

60

Experimental
title*The amount of both light chain and heavy chain were almost the samein before and after optimization. Therefore, the improvements formaking whole IgG were “solubilization” and “tetramer formation”.

ReducedSDS-PAGE

250
150

100

12

chaperone-like activity to β-barrel protein.*1

3. Synthesis of other IgGs using PUREfrex®

250

Step 3: Protein enrichment(AmiconUltra-0.5, MWCO: 30 kDa, Merck Millipore)100

isomerase (PPIase) from E. coli, which has achaperone-like function independent of PPIase
activity.*1

80

IgG

0

LC:HC

150

FkpA: Periplasmic peptidyl-prolyl cis-transSummary of the optimization

250

50

Light chain(23.6 kDa)

100

[kDa]

Step 2: Gel filtration(Superdex-200 10/300 GL, GE healthcare)150

Skp: Periplasmic protein from E. coli, which shows

DnaK mix

[kDa]

200

32

Purified IgG (Step 3)

Step 1: Protein A purification(KANEKA KanCapA™, Kaneka)

Protein A eluate(Synthesized product)chaperones from E. coli, which contains
5 µM DnaK, 1 µM DnaJ, 1 µM GrpE.*2

GSSG [mM/2 mM GSH]

50

Light chain

no DNA
Synthesized product1 mL of optimized PUREfrex 2.0 reaction mixture

250

37

2. Evaluation of the synthesized Trastuzumab
Non-reduced
SDS-PAGE

DnaK mix: Mixture of cytoplasmic molecular

100

100

300

Time [h]Purification method

1075

Heavy chain(50.6 kDa)

0
4

8

Non-reduced
SDS-PAGE

100

IgG

6

After
optimization

[kDa]

0

60

[kDa]

0
Heavy chain

GSH TCEP MPD 2-ME noreductant

100

Synthesized protein [µg/mL]

Synthesized protein [µg/mL]

50

DTT

Beforeoptimization

LC:HC

37℃
37℃

100

0

80

#1 and #9: Before and After optimization

IgG

Time [h]

150

20

100

TCEP: Tris(2-carboxyethyl) phosphine, 2-ME: 2-mercaptoethanol,
MPD: 3-Mercapto-1,2-propanediol#8: Template DNA ratio(Light chain/Heavy chain)

200

250
200

20

DsbC [µM]#7: Incubation time
30℃
30℃

>5 µM

20

40

sup
ppt

The DNAs of E. coli codon-optimized Trastuzumab were designed from amino acid sequences registered inpublic database (Drug Bank, https://www.drugbank.ca). To enhance the protein productivity, we changed
the codons of N-terminal 2nd to 6th amino acid to AT-rich codons (not “most frequently used codons”).
The synthetic genes were amplified with PCR, then purified and used to protein synthesis. The final 10 nM
of mixed template DNAs (light chain + heavy chain) were added to PUREfrex 2.0 reaction mixture.

30

60

total

Stop more than 10 any nucleotides

40

[kDa]

100

80

no DNA...TAATGAATAACTAATCC-3’

50

sup
ppt

FLAG

CH3

total

CH2

no DNA(λ1, 1488 bp, 462 aa)

CH1

E. coli, which promotes the correct disulfide bondformation.*1

150

100

Synthesized protein [µg/mL]

VH

Synthesized protein [µg/mL]

Heavy chain DNA

Synthesized protein [µg/mL]

150

CL

DsbC: Periplasmic disulfide bond isomerase from

250

150

400

200

250
200

300

Synthesized protein [µg/mL]VL(κ, 747 bp, 215 aa)

Light chain

250

150

Light chain DNA

Skp

[kDa]

Synthesized protein [µg/mL]

T7 promoter
5’-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATG-②③④⑤⑥②③④⑤⑥・・・②③④⑤⑥
Met AT-rich codons
SD sequence

FkpA

25 μM
50 μM

GSSG [mM/2 mM GSH]

DnaK
mix

50 μM

DsbC [µM]#5: Molecular chaperone and chaperone-like proteins×1 mix
25 μM#4: GSH/GSSG ratio

×0.5 mix#3: Reducing agent

no-Chaperones#2: DsbC concentration

Synthesized protein [µg/mL]

Template DNA design of Trastuzumab (PCR product)a supplement for the synthesis of aggregate-prone proteins

150

100
150
100

200
100

50
100

50

50
0

0

0

0

200

150

300

100

200

100

LC>HC

200

LC>HC

LC<HC

LC<HC

150

150

100

100

50

50

0

0

50

0

0

LC:HC
Yield

Light chain(23.6 kDa)

37

25

[%]

131

0.88

1.0

131

100

678

Purified IgG (Step 3)

0.3

4.57

99

68

0.1

15

75.4
Non-reduced SDS-PAGE on 10% gel.
CBB staining.
Samples: 1.0 μL/lane.
Trastuzumab: 500 ng/lane.

51.7

Non-reduced
SDS-PAGE

2.9

ELISA

Reduced SDS-PAGE on 12.5% gel.
CBB staining.
Purified IgG: 1.0 μL/lane.
Trastuzumab: 500 ng/lane.

Adalimumab

Association

Internalization analysis

1.9

EC50
0.9

Purified IgG

Trastuzumab

anti-human IgG

Recyclingendosome[kDa]

37°°C 3H

150

100
Purified IgG

Trastuzumab

CD63

-0.05
60

120

180

240

300

360

420

480

Time [sec]Purified IgG
Trastuzumab

37°°C 3H

KD [M]

kon [1/Ms]

koff [1/s]

4.24E-10

6.38E+05

2.70E-04

4.03E-10

6.29E+05

Late
endosome

2.53E-04

System: Octet RED96 System (Pall ForteBio)
Biosensor: Anti-Human IgG Fc Capture (AHC) biosensor(Pall ForteBio)
Buffer: Kinetics Buffer 10X (Pall ForteBio)
Ligand: “Purified IgG” or “Trastuzumab”
Analyte: 17.6, 8.8, 4.4, 2.2 and 1.1 nM of Extracellular domainof Human ErbB2/HER2 (Sino Biological)
KD: Measured affinity of interaction; affinity constant in Molar.
kon: Association rate constant. koff: Dissociation rate constant.

Lysosome

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
10 nM of “purified IgG” or “Trastuzumab”.
Cells were washed five times with thebinding medium and incubated at 37°C for
3h. Cells were then fixed and processed fordual-label indirect immunofluorescencemicroscopy. CD63 (Lamp3) is marker of lateendosomes and lysosomes. White arrows
indicate partial co-localization of anti-HER2IgG with CD63. Bar indicates 20 μm.

Non-reduced SDS-PAGE on 10% gel, Oriole staining.
Samples: 1.0 μL of reaction/lane.

EC50
0.9

-0.1

Concentration [nM]

Concentration [nM]

Concentration [nM]

IgG name

Subclass

AntigenMW [kDa]

Synthesis
temperatureTemplate DNA ratio(LC:HC, total 10 nM)

IgG Yield [µg/mL]

EC50 [nM]

Trastuzumab

IgG1κ

HER2

148.3

37°°C

1:2-1:4

124±
±9

0.16

Adalimumab

IgG1κ

TNF-α

147.4

37°°C

1:2-1:4

46±
±6

0.1

Cetuximab

IgG1κ

EGFR

148.3

30°°C

2:1

49±
±6

0.02

Panitumumab

IgG2κ

EGFR

144.9

37°°C

2:1

33±
±3

0.036

Nivolumab

IgG4κ

PD-1

144.2

30°°C

1:2

73±
±2

0.05

Yields are expressed as means ± SD (n=3). EC50: 50% effective concentration of target binding activity in ELISA.

anti-CD63

0

0.9

-0.1

250

0.00

Merge + DAPI

Binding [nm]

0.05

EC50

1.9

Antigen: PD-1,
Fc Fusion(20 ng/well)

Summary of the synthesis of several IgGs using PUREfrex®

0H

Early
endosome

0.9

2.9

Antigen: Human
EGFR/HER1
1.9 (12.5 ng/well)

2nd Ab: anti-human Fab 2nd antibody (Jackson Immuno Research), EC50: 50% effective concentration of target binding activity in ELISA.

HER2

0.10

EC50

-0.1

37°°C 0H

0.15

1.9

Nivolumab

2.9

Antigen: Human
TNF-α(40 ng/well)

Concentration [nM]Anti-HER2-IgG

Dissociation

0.20

IgG

Cetuximab & Panitumumab

2.9

Antigen: Human
ErbB2/HER2(1 ng/well)-0.1

Binding kinetics analysis

Heavy chain

Abs at 450 nm[µg]

Abs at 450 nm

[mL]

2.22

Light chain

IgG

Trastuzumab

[μM]

330

Heavy chain

20[µg/mL]25

Protein A eluate (Step 1)

Light chain

Abs at 450 nm

Synthesized productVolumeTotal
protein

Abs at 450 nm

IgG
concentration

Method for the synthesis of IgG using PUREfrex
37°°C 3H

The performance of purified IgG was
similar to the commercial Trastuzumab!!!

【Acknowledgements】】
We thank Dr. Yosuke Uchinashi (Nihon Pall Ltd.) and Keita Iguchi (KANEKA Corporation) for technical supportand experimental advice.

Conclusion
Coming soon(without DTT and with 2 mM GSH)
Reaction mix:++

(3 mM GSSG, 5 μM (117 µg/mL) DsbC)(5 μM DnaK, 1 μM DnaJ, 1 μM GrpE)

Incubation :30 - 37°°C for 28h

Template DNA:PCR product (final 10 nM mixed template DNA)

Synthesis temperature and template DNA ratio (light chain/heavy chain)should be optimized for individual IgGs for the best yield.
*If you want to synthesize multiple IgGs at the same time(e.g. IgG screening), we recommend “30°°C and LC:HC=1:1” as a first step.

・Aglycosylated IgG (including IgG1, IgG2 and IgG4 subclasses) was
synthesized in the active form using PUREfrex 2.0.
・Disulfide bond isomerase (DsbC), redox state (reducing agent/oxidizing
agent ratio) and long-time incubation were important to form acorrect hetero tetramer of two heavy chains and two light chains.
・IgG yield, optimum synthesis temperature and optimum template DNA
ratio were different between individual IgGs, which may reflect thedifference of CDR sequence and structural stability.
・PUREfrex® will be “useful” and “powerful” tool for high-throughput
production/screening of functional antibodies.