Efficient in vitro expression of aglycosylated full-length IgG usinga reconstituted cell-free protein synthesis system, PUREfrex® 2.0○Satoshi Murakami, Rena Matsumoto, Takashi Kanamori
GeneFrontier Corporation (Chiba, Japan)

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

Abstract: Aglycosylated full-length IgG (anti-HER2 monoclonal antibody) was synthesized using PUREfrex 2.0. The PURE (Protein synthesis Using Recombinant Elements) system is a reconstituted cell-free protein synthesis system, which consists of only purified factors
necessary for transcription, translation and 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 functional proteins such as
Fab, scFv, a protein based toxin and an immunotoxin last year. Here, we report the further application using PUREfrex 2.0 for production of IgG. To synthesize IgG, we optimized the composition of the reaction mixture and the reaction conditions as below; 1) adding molecularchaperone (DnaK mix) to increase the solubility of the product; 2) adding DsbC to form disulfide bonds between the correct cysteine residues; 3) adjusting GSH/GSSG ratio for optimum redox state; 4) long-time incubation (over 20h) to assemble the hetero tetramer of two heavy
chains and two light chains. At the best mode of synthesis, the productivity of IgG reached to 0.042 mg/mL. The synthesized IgG was detected as single band on non-reduced SDS-PAGE after the purification by protein A resin and the following gel filtration. The purified IgG exhibitedhigh binding affinity to recombinant HER2 protein. Fifty percent effective concentration of target binding activity (EC50) in ELISA was 0.16 nM, which is similar to the trastuzumab. This result indicates that PUREfrex 2.0 will be useful tool for high-throughput expression/screening offunctional antibodies (scFv, Fab, and IgG).

2. How to use PUREfrex®; “Easy” and “High throughput” preparation of Proteins

1. PUREfrex®; based on the PURE system technology

Template DNA
E. coli based cell-free protein synthesis systems

ORFT7 promoter

DNA

Extract system
Ribosome

ortRNAs

Typical Yield (μg/mL)

NTPs

T7 terminator

Reconstituted system

Amino Acids

Translation Factors

T7 promoter
5’-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCAATG-②③④⑤⑥②③④⑤⑥・・・②③④⑤⑥
MetSD sequence

S30 system

PURE system(original*)

100-1,000

10-200or10-200! To enhance the protein productivity,
we recommend to change the codons ofN-terminal 2-6 amino acids to AT-richcodons (not “most frequently usedcodons”).

ORF

20-1,000...TAATGAATAACTAATCCContamination
RNase

very HighLow

very Lowvery Low

Endotoxin (LPS)very HighHighvery Lowvery Low

Plasmid DNA

OKOKOKOK

PCR product

NGOKOKOK

Customization ofReagent

Difficult

Easy

Easy

Easy

OKNG

OKOK

Stop more than 10 any nucleotides

T7 promoter-driven protein
expression vector for E. coli

PCR product containing T7 promoter and SD sequence

Template DNA

Purification ofHis-tagged product

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

Use forApplication(Purification, if necessary)

In vitro display
Ribosome display△○◎○mRNA display

△○

◎

○

① Mix solution I,II and III【Reference】 *: Shimizu Y. et al. (2001) Nat. Biotechnol., vol. 19, p. 751② Addition oftemplate DNA③ Incubation at
37°°C for 2-4 hours

Total 2-4 hours

3. Optimization of the synthesis of full length IgG
GSH/GSSG ratio

DsbC concentration
No.

12345No.

Full length IgG

DsbC
Full length IgG

42.4

20.0

10.0

18.0

75%
50%25%

DTT*1(2 mM)

No. 1

No. 2

No. 3

No. 4

No. 5

13579

GSH [mM]

GSH*2(3 mM)

GSSG [mM]

1086459117235

469 [µg/mL]

1.25

2.5

5.0

1020 [µM]
Light chain

100%
75%
50%

123456789Time course of synthesis
010

Full length IgG

Full length IgG

100%0%0.0

Full length IgG

29Synthesized protein [%]30.0

Heavy chain

Synthesized full length IgG [%]40.0

Synthesized protein [%]Synthesized full length IgG[µg/mL]

Light chainMolecular chaperone and chaperone-like proteins

Heavy chain

Full length IgG

75%
50%
25%

25%

No.1

51015

No. 2

No. 3

No. 4

No. 5

No. 6

No. 7

No. 8

No. 9

6.5

13.0

26.1

52.2

7.3

14.6

29.3

58.5

20

No. 1050%Light chain

25%

Heavy chain

37→30°°C

FkpA [µM]

Full length IgG

0%Skp [µM]

0DsbC [µM]

212 16 20 24 28 32 [H]

75%

0%
08

100%

100%0%

4

Full length IgG

Synthesized protein [%]Reducing agent(DTT or GSH)

48121620242832Time [H]FkpA: Periplasmic peptidyl-prolyl cis-trans isomerase (PPIase) from E. coli, which has a chaperone-like function independent of PPIase activity.*1
Skp: Periplasmic protein from E. coli, which shows chaperone-like activity to β-barrel protein.*1
DnaK mix: Mixture of cytoplasmic molecular chaperones from E. coli, which contains 5 µM DnaK, 1 µM DnaJ, 1 µM GrpE.*2
DsbC: Periplasmic disulfide bond isomerase from E. coli, which promotes the correct disulfide bond formation.*1【Reference】 *1: Goemans C. et al. (2014) BBA. vol. 1843, p. 1517 *2: Castanié-Cornet MP. et al. (2014) BBA. vol 1843, p1442

Aglycosylated full-length IgG (anti-HER2 monoclonal antibody) was synthesized using PUREfrex 2.0 under various conditions. After synthesis, all samples were centrifuged at 9,100 xg for 10 min and1.0 µL of supernatant was applied to non-reduced (10% gel) or reduced (10-20% gel) SDS-PAGE. The gels were stained with Oriole fluorescent gel stain (Bio-Rad) and the protein bands were quantitatedusing LAS-4000 system (GE Healthcare).
*1: Initial condition of the optimization (PUREfrex 2.0 (normal version, 2 mM DTT) with 3 mM GSSG, 5 µM DsbC and DnaK mix; Reported at PepTalk 2016, San Diego, CA, USA).
*2: Final condition of the optimization. The yield of full length IgG was reached to 0.042 mg/mL.

Step 1: Protein A purification
Protein A cellulose (KANEKA KanCapA™, Kaneka)・Equilibrate with “wash buffer 2” and apply 25 µL of resin to
1 mL of PUREfrex reaction mixture.
・Rotate at 4°C for 1h.

Resin(without DTT and with 3 mM GSH)
Reaction mix:+(8 mM GSSG, 5 μM (117 µg/mL) DsbC)

250

Wash
buffer 220 mM Na-Phosphate buffer (pH 7.0), 0.05% Tween 20
・Add 500 µL to resin. Incubate at 4°C for 2 min. 3 times.

100Elutionbuffer

50 mM Glycine-HCl (pH 2.5), 0.05% Tween 20
・Add 100 µL to resin. Incubate at 25°C for 3 min. 3 times.
・Add 5 µL of 1 M Tris-HCl (pH 8.8) to the eluate for adjusting
to neutral pH.

Wash
buffer 1CH1**

5 nM++5 nMColumn

Superdex-200 10/300 GL (GE healthcare)

Buffer

PBS-T (0.05% Tween 20)

Flow rate

0.5 mL/min (using ÄKTA system)

Elution position

37°°C for 4h to 30°°C for 16h (total: 20 h)

75

Light chain(23.6 kDa)

AmiconUltra-0.5 (MWCO: 30 kDa, Merck Millipore)
Concentrate 2 mL of the eluate to 100 µL.

Trastuzumab

Dissociation

2.4

0.10
0.05

Trastuzumab

60

120

180

240

300

360

420480Merge + DAPI

anti-CD6337°°C 3H

Our Products:

34.3

0.23

1.0

34.3

10037

Protein A eluate(Step 1)

103.6

0.70

0.3

31.1

90.6

2520

Purified IgG(Step 3)

157.0

1.06

0.1

15.7

45.7Purified IgG
Trastuzumab
anti-HER2-Fab(produced by E. coli)

1.9

EC50

1.4

EC50 = 0.16 nM
0.90.4

KD [M]

kon [1/Ms]

koff [1/s]

Purified IgG

4.03E-10 ±1.0E-11

6.29E+05 ±4.7E+03

2.53E-04 ±6.0E-06

Trastuzumab

4.24E-10 ±8.8E-12

6.38E+05 ±4.2E+03

2.70E-04 ±5.3E-06

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 domain ofHuman ErbB2/HER2 (Sino Biological)
KD: Measured affinity of interaction; affinity constant in Molar.
kon: Association rate constant. koff: Dissociation rate constant.

VLVH

CL

CH1

・The 15.7 µg of synthesized IgG was purified
from 1.0 mL of PUREfrex 2.0 reaction mixture.

On-going Project-0.1
0.001

0.01

0.1

1

10

100

Concentration [nM]
Antigen: Extracellular domain of Human ErbB2/HER2 (1 ng/well)
2nd Ab: anti-human Fab 2nd antibody (Jackson Immuno Research)
EC50: 50% effective concentration of target binding activity.

The performance of purified IgG
was similar to the trastuzumab!!!

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

・Redox state (GSH/GSSG ratio) and long-time
incubation (over 20h) were important to forma correct hetero tetramer of two heavy chains
and two light chains.

EC50 = 1.8 nM

Time [sec]37°°C 3H

Synthesized
product

2.9Abs at 450 nm

Binding [nm]anti-human IgG

Association0.20

0BT-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 withthe binding medium and incubated at 37°C for 3h.
Cells were then fixed and processed for dual-label
indirect immunofluorescence microscopy. CD63
(Lamp3) is marker of late endosomes and lysosomes.
White arrows indicate partial co-localization of antiHER2-IgG with CD63. Bar indicates 20 μm.

[%]

・Aglycosylated full-length IgG was synthesizedin the active form using PUREfrex 2.0.

-0.05

Lysosome

[µg]

ELISA

Purified IgG

Lateendosome[mL]

Reduced SDS-PAGE on 10-20% gel (CBB staining).
Purified IgG: 3.0 μL/lane.
Trastuzumab: 450 ng/lane.

Non-reduced SDS-PAGE on 10% gel (CBB staining).
Samples: 1.0 μL/lane.
Trastuzumab: 200 ng/lane. M: marker.

0.00

CD63

[μM]

10Full length IgG: 21-25 min (total: 2 mL)

0.15

37°°C 3H[µg/mL]

Conclusion

37°°C 0H0H

Yield

15Binding kinetics analysis

HER2Totalprotein

Step 3: Protein enrichment

Internalization analysis
Anti-HER2-IgG

Volume5037

5. Evaluation of purified full length IgG

Recyclingendosome

Heavy chain(50.6 kDa)

50IgG concentration

250
150
10075(148 kDa)

Purification summary

25Centrifugal filter

Purified IgG

[kDa]

CH3

Step 2: Gel filtration*We recommend to change the codons of N-terminal 2-6 amino acids
to AT-rich codons (not “most frequently used codons”) to enhance theprotein productivity.

ReducedSDS-PAGE

CH2150PCR product (final 10 nM mixed template DNA)HC DNA

Earlyendosome

VHVL

20 mM Na-Phosphate buffer (pH 7.0), 20 mM MgOAc,
0.05% Tween 20・Add 500 µL to resin. Incubate at 4°C for 2 min.

(5 μM DnaK, 1 μM DnaJ, 1 μM GrpE)LC DNA

Incubation:

M

Full length IgG

CL+Template DNA:

[kDa]

Purified IgG (Step 3)
Trastuzumab

Optimized condition of IgG synthesis

Non-reduced
SDS-PAGE

Trastuzumab

Purification method

no DNA
Synthesized productProtein A eluate
Purified IgG (Step 3)

4. Synthesis and purification of full length IgG using PUREfrex® 2.0・Synthesis of other IgGs.
(e.g. anti-TNF-α, EGFR, VEGF, etc.)
・Synthesis of bispecific IgG antibody.
【Acknowledgements】】
We thank Dr. Yosuke Uchinashi (Nihon Pall Ltd.) andKeita Iguchi (KANEKA Corporation) for technical supportand experimental advice.

/a supplement for the synthesis of proteins containing disulfide bonds

a supplement for the synthesis of aggregate-prone proteins