ヒトプロテインジスルフィドイソメラーゼを添加した®
再構成型無細胞タンパク質合成系(PUREfrex )によるジスルフィド結合タンパク質合成
Synthesis of proteins containing disulfide bonds using PUREfrex® with human protein disulfide isomerase○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)

truncated version oftissue plasminogen activator (tPA)

Gaussia Luciferase (GLuc)

Homo sapiens
36Ser-40Ile/211Gly-562Pro (+FLAG)
368 a.a.
41,072 Da
9

Experimental condition of protein synthesis(PDB ID: 1PK2/1BDA)

3

Incubation time

5

DsbC

hPDI + hEro1α

3

DsbC(-)

30℃,
24 h

4GSSG

(-)

10 µM

+ 0.5 mM Cys+ 4 mM GSH+ DnaK mix

422 mM

PUREfrex®2.1hPDI + hEro1α
conc.

30℃,
24 h

0-20 µM

0-8 mM

10 µM

5 μM DnaK
1 μM DnaJ,
1 μM GrpE(-)

0-2 µM

2 mM(-)(-)

0.125 µM

0 mM/2 mM

(-)

10 µM
(-)

(-)

TemplateDNA

PCR
product(1 ng/µL)

(-)

30℃,
24 h(-)

30℃,
0-24 h

20 µM

30℃,
24 h

Reducedslope: 0-14 min(1000 x Abs. 405 nm)

25(kDa)

20250
15015

51015
PDI conc (µM)

20+ 0.5 mM Cys+ 4 mM GSH

20 µM(-)(-)

20 µM(-)

1 mM(-)(-)

PCRproduct(1 ng/µL)

37℃,
4h・ SDS-PAGE (0.25 – 0.5 μL of reaction mix/lane) → Western Blotting (anti-FLAG) or Gel staining (Oriole)・ GLuc activity assay: BioLux Gaussia Luciferase Assay Kit (NEB#E3300S) (2 µL PURE reaction mix/50 µL
assay soln.)Non-reduced

GLuc activity
2400000
2000000
RLU

1600000
1200000(kDa)

80000010075

20102550

37redoxi25Non-reducedtPA activity

4 mM GSHtPAGSSG conc.
(mM)

246812500.5

Reduced3020(kDa)
250
150

5000246810redoxi

37

GSSG conc. (mM)

1-2 mM GSSG was required for 10 µM
hPDI in the synthesis of active tPA.

ReducedAcid phosphatase (AppA)

No.

100
75

515Escherichia coli
23Gln-432Leu (+FLAG)
421 a.a.
45,959 Da
4(PDB ID: 1DKM)

Experimental condition of protein synthesis*10Combination of hPDI and Ero1α was also effective foractive GLuc synthesis.
DsbC rather inhibited the activity of synthesized GLuc.

Organism
Synthesized region
LengthMolecular weight
No. of disulfide bonds

4-2. hPDI + GSSG concentration

15redoxi20(*polymerized and stacked)

slope: 0-14 min(1000 x Abs. 405 nm)

(-)

2525

More than 10 µM hPDI (with 2 mM GSSG)
was required for synthesis of active tPA.

in Endoplasmic Reticulum (ER)0.125 µM

3750
0

2. Oxidative folding of nascent polypeptide

(-)

10075

10Reductant (DTT) and Cysteine

It enables to select a suitable reducing agent forthe protein of your interest.

10 µMTemp.
& Time300=PUREfrex®2.1*5-(-)

035(Ref: Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751.)(-)

Template
DNAtPA0DNA(-)

2010251400・Low level of contamination ・High throughput preparationof proteins
・Easy adjustment of thereagent composition(including Fab, scFv,
protein toxin etc.)
・PCR products usable as atemplate DNA
・Protein science research・in vitro display (Ribosomedisplay, mRNA display etc.)

PDI conc.
(µM)

(-)

400000tPADNA(-)

tPA activity

10 µM

Non-reduced

14-1. hPDI concentration

ApplicationDsbC

DNA(-)

2

hPDI + GSSG
conc.

(-)

DsbC

hEro1αGSSG

hPDI conc.

hPDI

hEro1αGSSG

DsbC

11GSSGhPDI

Basic condition

Temp.
& TimehPDI

Basic condition

FactorshEro1αFactorsNo.

hPDI + hEro1αExperimental conditions of protein synthesis
No.

Gaussia princeps
18Lys-185Asp (+FLAG-His6)
187 a.a.
20,407 Da
5hPDI

Organism
Synthesized region
LengthMolecular weight
No. of disulfide bonds

Organism
Synthesized region
LengthMolecular weight
No. of disulfide bonds

・ SDS-PAGE (0.25 – 0.5 μL of reaction mix/lane) → Western Blotting (anti-FLAG) or Gel staining (Oriole)・ tPA activity assay: Assay of chromogenic substrate cleavage activity (2.5 µL PURE reaction mix/ 200 µL assay soln.)

1. PUREfrex®; based on the PURE system technology
Advantage

5. Result 3: Examples of other disulfide bonded
protein synthesis

Isomerase(-)

PURE systemは、大腸菌のタンパク質合成に関与する因子のみから再構成した無細胞タンパク質合成系である。PURE systemにプロテインジスルフィドイソメラーゼ(PDI) として大腸菌DsbCを添加することで、活性を有したジスルフィド結合タンパク質を合成できる。複雑な構造のタンパク質や高等生物のジスルフィド結合タンパク質合成に広く対応するため、本発表ではPUREfrex® (改良型PURE system) を用い、DsbCに替えてヒトPDI(hPDI) およびその関連タンパク質を利用できるか検討した。モデル系として、9つのジスルフィド結合を持つ組織プラスミノーゲン活性化因子 (tPA)の合成を行った。還元剤に還元型グルタチオン (GSH) を使用したPUREfrexをベースに、酸化型グルタチオン (GSSG) 存在下でhPDIを添加してtPAを合成すると、合成されたtPAはhPDIの添加濃度依存的に活性が増加した。一方、
GSSG非存在下でも、hPDIおよびhEro1α (PDI酸化酵素 )を添加しtPAを合成した場合、合成されたtPAは同様に高い活性を示した。すなわち、hPDIを機能させるためにはGSSGあるいはhEro1αの添加が必要であることが分かった。また、GSSG存在下でhPDIとDsbCの添加効果を比較したところ、hPDIはDsbCとほぼ同等の効果を示した。以上より、hPDI添加でも、酸化還元環境の調節やhPDI酸化酵素の添加により効率よくジスルフィド結合タンパク質の合成が可能であることが確認された。さらに他の基質タンパク質の合成についても報告する。

4. Result 2: Disulfide bonded protein synthesis
using hPDI and hEro1α with PUREfrex®

Reduced<Abstract>

hPDI

GSSG

hEro1α

DsbC

1hPDI

2 µM

2 mM(-)(-)

2

hPDI + GSSG

2 µM

0 / 2 mM(-)(-)

3hPDI + hEro1α

PUREfrex®2.12 µM(-)

0.2 µM(-)

4DsbC + GSSG

+ 0.5 mM Cys+ 4 mM GSH(-)

0 / 2 mM

(-)

4 µM

0 / 2 µM

0 / 2 mM

0 / 0.2 µM

0 /4 µM

DnaK mix
5 µM DnaK1 µM DnaJ
1 µM GrpE

525Basiccondition

Factors(*polymerized and stacked)

Template
DNA

Temp. &TimePCRproduct(1 ng/µL)

37℃, 4 h・ SDS-PAGE (0.5 μL of reaction mix/lane) → Western Blotting (anti-FLAG)・ AppA activity assay: Assay of PNPP dephosphorylation activity (1 µL PURE reaction mix/100 µL assay soln.)

4-3. hPDI + hEro1α concentration

Non-reduced

AppA activity(kDa)

15250
150

101007552-1. PDIs and related protein used in this study+15300
10200

51008121620
Incubation time (h)(PDB ID: 1AJA)

2 mM DTT(-)(-)

10 µM(-)

0.2 µM(-)

2 mM(-)

TemplateDNA

Synthesized protein

plasmid(10 ng/µL)

PUREfrex®2.1 supplemented with hPDI in the presence ofGSSG or hPDI oxidase (hEro1α) can synthesize disulfide
bonded proteins with their activities.
GSSG is unnecessary when oxidizing hPDI is applied withhEro1α.

28Activity

8624DNA(-)

10 µM hPDI + 0.125 µM hEro1α*・Necessary conditions for the synthesis of disulfide bondedproteins with activities are different for each proteins.
Protein name

Basic reaction
mix

37℃, 4 hredoxi

Alkaline
Phosphatase(AP)

redoxiIsomerase

PUREfrex®2.1(+ 0.5 mM Cys)

Oxidant/Oxidase

2 mM GSSG
2 mM DTT

25
Trancatedversion ofTissure
Plasminogen
Activator (tPA)

Reduced4-5. Comparison of hPDI with DsbC in tPA synthesis

Gaussia
Luciferase(GLuc)

tPA activity
slope: 0-14 min(1000 x Abs. 405 nm)

30

Non-reduced

25
20
15
10AcidPhosphatase(AppA)

5*DNA(-)(+ 0.5 mM Cys)
+ DnaK mix

PUREfrex®2.1(+ 0.5 mM Cys)

PUREfrex®2.1(+ 0.5 mM Cys)

4 mM GSH

0.125 µM hEro1α

10 µM hPDI

2 mM GSSG

10 µM hPDI

0.125 µM hEro1α

20 µM DsbC

2 mM GSSG
1 mM GSSG

4 mM GSH

4 mM GSH

10 µM hPDI

0.125 µM hEro1α

2 µM hPDI

2 mM GSSG

2 µM hPDI

0.2 µM hEro1α

4 µM DsbC

2 mM GSSG

In PUREfrex®2.1, it is possible to select a suitable
isomerase (hPDI etc.) and redox environment dependingon the target protein.

10075

2 mM DTT

redoxi50

4 mM GSH

redoxi37redoxi

Combination of hPDI and GSSG or hEro1α showedalmost the same effect as the combination ofDsbC and GSSG.

PUREfrex®2.1

10 µM hPDI(kDa)
250
150

GSH was a more suitable reducing agent forforming the disulfide bond of the proteins than DTT.

Reductant
4 mM GSH

0GSSG

hPDI + hEro1αhPDI

Isomerase(-)

24

Non-reduced

424DNA(-)

86421Time (h)8121620
Incubation time (h)(*polymerized and stacked)

Non-reducedDNA(-)GSSG

hPDI + hEro1α

4ActivitytPAorActive disulfide bonded protein

50Temp. &Time+002

Reduced: 24 h00DNA(-)

10 µM

hPDI

slope:5-30 min(1000 x Abs. 405 nm)

5100

DsbC + GSSG

hEro1αReduced

2 mM
GSSG

10200

hPDI + hEro1α
DsbC

GSSG

Isomerase(-)
4 mM GSH

10 µM
hPDI +0.125 µM
hEro1α300

28

Non-reduced

tPA

15

Further incubation increased
the activity of the synthesized
tPA.

37

hPDI

AP activity

10 µM
hPDI

400(kDa)
250
150
10075

3-1. Difference in synthesized AP activity
by reducing agent

Isomerase(-)

20*・ SDS-PAGE (0.5 μL of reaction mix/lane) → Gel staining (Oriole)・ AP activity assay: Assay of PNPP dephosphorylation activity (0.1 µL PURE reaction mix/100 µL assay soln.)

161412108642024

Reduced: 24 h

410 µM hPDI + 2 mM GSSG

Time (h)50000hPDI

hEro1α(- GSSG)

slope: 0-14 min(1000 x Abs. 405 nm)

400

Synthesized protein(ng/µL reaction)

2025

hPDI + GSSG

GSSG

+ 0.5 mM Cys・hPDI can be used as an isomerase with PUREfrex®2.1.

600

hPDI

3PUREfrex®2.1

hPDI

Isomerase(-)

hPDI + hEro1α

4 mM GSH
or2 mM DTT

hEro1α+ (- GSSG)Reduced2(*polymerized and stacked)

slope: 0-14 min(1000 x Abs. 405 nm)

500

ReducedhPDI

Isomerases(-): Isomerase (-)
C: 4 µM DsbC
P: 2 µM hPDI
E: 0.2 µM hEro1αtPA synthesis and activity(10 µM PDI + 0.125 µM Ero1α)0Experimental conditions of protein synthesis

137+1

P0AEG6Escherichia coli
217 a.a.
23,591 Da

Escherichia coli
22Arg-471Lys451 a.a.
47,330 Da
2ReductantredoxiGSSG0(PDB ID: 3AHQ)

Alkaline phosphatase (AP)Factors

NRNR(-) C PP+E(-) C PP+E

Combination of hPDI and Ero1α was also effective for active AppA synthesis.
Addition of DnaK mix was also efficient for the synthesis of active AppA.

25=25

3. Result 1: Selection of suitable reducing agent

No.

GSSG

600

Synthesized protein

Basiccondition

DnaK (+)

1.0

4-4. Incubation time

DsbC (thiol:disulfide interchange protein)

Organism
Synthesized region
LengthMolecular weight
No. of disulfide bonds(kDa)50

2.0NRNR(-) C PP+E(-) C PP+E

DnaK (-)

oxitPA synthesis and activity(10 µM PDI + 2 mM GSSG)

UniProt ID
Organism
LengthMolecular weight

3.0

DnaK (+)

6. SummaryhPDI

Q96HE7
Homo sapiens
446 a.a.
52,122 Da(PDB ID: 1EEJ)

50

2.5

P07237
Homo sapiens
492 a.a.
55,424 Da

hEro1α (human endoplasmic oxidereductin)
UniProt ID
Organism
LengthMolecular weight2

hEro1α oxidized hPDI in the absence ofGSSG and as a result was able tosynthesize active tPA.

Synthesized protein(ng/µL reaction)(PDB ID: 4EKZ)

11.5hEro1α conc. (µM)

DnaK (-)

4 mM GSH 4 mM GSH 4 mM GSH 4 mM GSH
GSSG(-)GSSG(-) 2 mM GSSG 2 mM GSSG
DnaK(-)
DnaK(+)
DnaK(-)
DnaK(+)
Isomerase (-)
4 µM DsbC
2 µM hPDI
2 µM hPDI + 0.2 µM hEro1α37

hPDI (human protein disulfide isomerase)
UniProt ID
Organism
LengthMolecular weight

0.5

4 mM GSH/ 2 mM GSSG

4.0

0.000

4 mM GSH/ 0 mM GSSG(-): R0.125 µM

20AppA(-): R

210.50.125

Ero1α conc.
(µM)

*25

AppA

5.0
slope: 5-20 min(1000 x Abs. 405 nm)

slope: 0-14 min(1000 x Abs. 405 nm)

300.031tPA activity0DNA(-)

tPA

25Reduced(*polymerized and stacked)

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