活性型タンパク質合成のための、再構成型無細胞タンパク質合成系(PUREfrex®)を用いたアプローチ
Investigation on how to synthesize active proteins by using a reconstituted cell-free protein synthesis system (PUREfrex®)○松本 令奈、金森 崇 (ジーンフロンティア株式会社)○ Rena Matsumoto and Takashi Kanamori (GeneFrontier Corporation)<Abstract>

1. PUREfrex®; Reconstituted cell-free protein synthesis system
based on the PURE system technology

PUREfrex® is a reconstituted cell-free protein synthesis kit developed basedon the PURE system. Now the productivity of PUREfrex® is up to 1 mg/mL.
However, depending on the protein, it may be difficult to synthesize asfunctional and soluble form by using only PUREfrex®.
In such cases, we can apply chaperones (DnaK and GroE) for correct
folding and isomerases (DsbC and PDI) for disulfide bond formation asadditives for PUREfrex®. Here, we introduce examples of protein synthesisthat could not be solved by these additives alone, but could be solved byadding other additives or synthesizing at lower temperature.
We mainly show examples of human acetylcholinesterase (hAChE)
synthesis. hAChE has three intramolecular and one intermolecular disulfidebonds. When hAChE was synthesized by using PUREfrex® with PDI, active
hAChE was obtained but there was a problem in solubility. The synthesis
under the presence of surfactant Brij 58 increased the solubility of hAChE
but decreased its activity. Next, we examined whether the difference in thesolubility of hAChE is depending on the synthesis temperature. The solubilityof the product was less than 10% at 30ºC although the amount of theproduct was high. In contrast, the product synthesized at 25ºC was almost
soluble although the amount was low. It was also found that
DnaK/DnaJ/GrpE are essential for the synthesis of soluble hAChE even atlower temperature.
This result indicates that it is possible to synthesize difficult-to-express
proteins by using PUREfrex® with optimization of conditions. In thispresentation, we also discuss the results from other types of proteins andadditives.

Advantage

Application

・Low level of contamination
・Easy adjustment of thereagent composition
・PCR products usable as atemplate DNA

2. Result 1: Influence of surfactant on protein
synthesis by PUREfrex®
Experimental conditions
for protein synthesis・High throughput preparationof proteins(including Fab, scFv,protein toxin etc.)
・Protein science research・in vitro display (ribosomedisplay, mRNA display etc.)

Reaction
mixtureIncubation

Template
DNA37ºC
4hsfGFP
PCRproduct(1 ng/µL)

PUREfrex®2.1(4 mM GSH)+
Sufractants(Ref: Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751.)→ Measurement of GFP fluorescence

For the synthesis of difficult-to-express protein…
Additives:・ Surfactants
・ Molecular Chaperones

Functional
proteinand / or+

Reaction conditions:・ Low temperature(20oC-25oC)• Most surfactants did
not inhibit the protein
synthesis reaction byPUREfrex® below theCMC.
• Some surfactants such
as Triton X-100 andTween 20 could be usedeven above the CMC.

3. Result 2: Effect of molecular chaperones on the solubilization of the synthesized product by PUREfrex®
3-1. Increasing the amount of DnaK Mix (DnaK/DnaJ/GrpE) facilitates
the solubilization of the products during protein synthesis.

3-2. DnaK Mix and ClpB can solubilize the aggregated products after protein synthesis.

Polyphosphate:AMP phosphotransferase (PPK2)

Luciferase

Experimental conditions for protein synthesis
Reaction mixture

PUREfrex®2.1(4 mM GSH)

FactorIncubation

Concentration of DnaK Mix
1x; 5 µM DnaK, 1 µM DnaJ, 1 µM GrpE

Template DNA30ºC
4h

PCR product(1 ng/µL)

・Measurement of solubilized products→ All samples were centrifuged at 20,000 xg for 30 min.
→ Synthesized protein and supernatant were applied to SDS-PAGE gel (12.5%).
→ Gel staining: Oriole Fluorescent Gel Stain (Bio-Rad)→ Quantitation: LAS-4000 system (GE Healthcare)OrganismSynthesized region
LengthMolecular weight500
400300

Photinus pyralis
1Met – 550Leu
550 a.a.
60,745 Da

Incubation

Template DNA

PUREfrex®2.1 (4 mM GSH)

37oC, 4 h

PCR product (2.5 ng/µL)

without molecular chaperones

PUREfrex® reaction mixture containing synthesized luciferase

200+ Chaperones:
DnaK Mix (5 µM DnaK/ 1 µM DnaJ/ 1 µM GrpE)
1 µM ClpB (E. coli)

1000DnaK
Mix (-)

1x
DnaKMix

・Luciferase activity assay
・Measurement of solubilized protein

2xDnaKMix

4Amount of Luciferase protein(ng/µL reaction)

Cytophaga hutchinsonii
1Met – 284Ser (+6xHis)
292 a.a.
33,921 Da

Reaction mixture

Luciferase activity:
relative RLU (vs. Chaperone(-))

600

Amount of PPK2 protein(ng/µL reaction)

Organism
Synthesized region
LengthMolecular weightExperimental conditions for protein synthesis

3210600
500
400
300
200
1000

synthesized protein
soluble protein

The solubility of the synthesized PPK2 increased by adding twice the amount ofDnaK Mix to the reaction mixture.

total protein

Aggregated synthesized luciferase could be solubilized by adding a combinationof DnaK Mix and ClpB to the reaction mixture after protein synthesis.

soluble protein

4. Result 3: Optimization of reaction conditions for functional protein synthesis by PUREfrex®
Human Acetylcholinesterase (hAChE)

Reaction mixture

Incubation time

Template DNAPUREfrex®2.1 (4 mM GSH)

24 h

PCR product(1 ng/µL)

10 µM hPDI/ 0.5 µM hEro1α4-3. It is possible to synthesize soluble and functional
hAChE by synthesis at low temperature.

4-1. Influence of surfactants for hAChE synthesis
Surfactant

DnaK Mix

TemperatureSurfactant

CHAPS/ Triton X-114/ Tween 20/ Brij 58

(+)

30oC

DnaK Mix

Temperature

Temperature(+)

20oC/ 25oC/ 30oC

malPEG2K(-) malPEG2K(+)

600

700

synthesized protein

20100hAChE was synthesized with soluble
form in the presence of Brij 58.
However, the activity of the synthesized
hAChE was reduced.

PUREfrex® reaction mixture
containing the synthesized hAChE+ Tween 20(final conc. : 0%/ 0.001%/ 0.01%/ 0.1%/ 1%)

hAChE activity per reaction(relative value)

4-2. The timing of adding surfactant is important for the activity ofthe product.

Addition of surfactant after synthesis
increased the activity of synthesized hAChE.

120100806040400300200100024 h

PCR product(1 ng/µL)

60↓
Purification by Co2+-resin (TALON Metal Affinity Resin (Clontech))(Binding buffer: 50 mM Tris-HCl (pH 8.0)/ 500 mM NaCl/
5 mM Imidazole/ 0.1% Tween 20)↓Activity assay

50403020
10020℃

25℃

synthesized protein

30℃

20℃

25℃

30℃
(kDa)

soluble protein

Most of hAChE synthesized at below 25ºC was soluble andhad its activity.

(kDa)

250

250

150

150

1004-4. DnaK Mix is necessary for hACHE synthesis
even under low temperature condition (25oC).

1007575hAChE
5050Factor

DnaK Mix

Temperature(-)/ (+)

o37

DnaK Mix

40070
synthesized protein
soluble protein

300
20010002560

2550

1640
3020100

DnaK Mix (-)

DnaK Mix (+)

37

25 C500

20025oC

E5500

anti-FLAG WB

1: Surfactant (-)
2: 1% CHAPS
3: 1% Triton X-114
4: 1% Tween 20
5: 1% Briji 58
6: DNA (-)

30hAChE activity assay

600

soluble protein

40(+)

E4redoxi

37

PUREfrex®2.1 (4 mM GSH)

E375

Template
DNAE2100

0Incubation
time

E1100Temperature

W2200

DnaK MixT(kDa)
250150hAChE activity per reaction(relative value)?

300

Reaction mixture

10 µM hPDI/ 0.5 µM hEro1α70hAChE activity per reaction(relative value)

400

Protein synthesis

1 2 3 4 5 6Amount of hAChE protein(ng/µL reaction)

1 2 3 4 5 6500

50hAChE activity per reaction(relative value)

Factor

4-5. Purification of synthesized hAChE

W1700Amount of hAChE protein(ng/µL reaction)

Amount of hAChE protein(ng/µL reaction)

Factor・Measurement of solubilized protein
・AChE activity assay: Amplite™ Colorimetric Acetylcholinesterase Assay Kit (AAT Bioquest).

FT

Homo sapiens
32Glu – 614Leu (+FLAG – 6xHis)
602 a.a.
66,812 Da
4 (intermolecular: 1)

DnaK Mix (-) DnaK Mix (+)

Low temperature (25oC) + DnaK Mix → Soluble hAChE

hAChE specific activity(per ng protein)

Organism
Synthesized region
LengthMolecular weight
No. of disulfide bonds

Basic experimental condition for protein synthesis

1412108

Highly active hAChE could
be purified from the reaction
mixture of PUREfrex®.

6420reaction
mixturepurifiedhAChE

5. Summary・Surfactants can be used for protein synthesis by PUREfrex®. However, some proteins require investigation in the timing of the use of surfactants.
・Increasing the amount of DnaK Mix improve the solubility of synthesized PPK2.
・Soluble hAChE is obtained by protein synthesis at lower temperature. DnaK Mix is essential for the soluble protein synthesis even at low temperature.
Optimal conditions for synthesizing proteins with PUREfrex® differ for each protein.
The great advantage of PUREfrex® is that it is possible to try various variations of additives and synthesis conditions.

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