再構成型無細胞タンパク質合成系 (PUREfrex®) を用いた糖タンパク質合成
Synthesis of glycosylated proteins using PUREfrex®
○松本 令奈1, 丹羽 達也2, 田口 英樹2, 金森 崇1(1ジーンフロンティア株式会社、2東工大・研究院・細胞センター)
PUREfrexは、PURE systemを基にした大腸菌で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。PUREfrexでは、ポリペプチドの合成のみが行われるが、適切な追加因子を使用することで翻訳後修飾も可能である。本発表では、糖転移酵素および基質となる糖供与体を用い、PUREfrexでN型糖鎖修飾タンパク質の合成を行った。糖鎖修飾反応はJewettらの方法を参考にした(Kinghtlinger et al.(2019))。初めに、大腸菌Colicin-E7 immunityproteinにN型糖鎖付加配列を挿入したモデルタンパク質(Im7-6)、ならびに数種類の糖転移酵素をPUREfrexでそれぞれ合成し、これらと糖供与体を混合してIm7-6の糖鎖修飾反応 (in vitro glycosylation (IVG)) を行った。反応後のSDS-PAGEおよび質量分析により、糖鎖付加配列中のアルギニン残基へのグルコース付加を起点とし、ガラクトース、GlcNAc、シアル酸の付加を確認した。さらに、Im7-6と糖転移酵素の合成および糖鎖修飾反応をonepotで同時に行ったところ、この方法でも糖鎖修飾されたIm7-6を確認できた。また、ジスルフィド結合を含むタンパク質や膜タンパク質に対しても、糖鎖付加が可能であることを確かめた。この結果より、PUREfrexでも糖タンパク質を合成できることが示された。

○ Rena Matsumoto, Tatsuya Niwa, Hideki Taguchi and Takashi Kanamori(1GeneFrontier Corp., 2Cell Biology Center, IIR, Tokyo Tech)

PUREfrex®
Translation process

Reconstituted cell-free protein
synthesis system based on the PUREsystem technologyPUREfrex®
Translation process

Polypeptides

Polypeptide ≠ Functional protein
Reconstituted cell-free protein
synthesis system based on the PURE
system technology

Synthesis of functional proteins using PUREfrex®
Translation process

Maturation process•Formation of higher-order structure•Folding of polypeptide•Formation of complex•Post-translational modification•Formation of disulfide bonds•Phosphorylation•Glycosylation•Lipidation•Processing•Localization
Reconstituted cell-free protein
synthesis system based on the PURE
system technology•Insertion into membrane•Translocation across membrane

Functionalproteins

Synthesis of functional proteins using PUREfrex®
Translation process

Maturation process•Formation of higher-order structure•Folding of polypeptide•Formation of complex•Post-translational modification•Formation of disulfide bonds•Phosphorylation•Glycosylation•Lipidation•Processing•Localization
Reconstituted cell-free protein
synthesis system based on the PURE
system technology•Insertion into membrane•Translocation across membrane

Functionalproteins

Synthesis of functional proteins using PUREfrex®
Translation process

Maturation process•Formation of higher-order structure•Folding of polypeptide•Formation of complex•Post-translational modification•Formation of disulfide bonds•Phosphorylation•Glycosylation•Lipidation•Processing•Localization
Reconstituted cell-free protein
synthesis system based on the PURE
system technology•Insertion into membrane•Translocation across membrane

Functionalproteinsin vitro Glycosylation (IVG) of target proteins combinedwith Cell-Free Protein synthesis (CFPS) by PUREfrex®
Glycosylation
Glc

Glycosyltransferase

Sugar donor

ApNGT (A. pleuropneumoniae)
ApNGT (A. pleuropneumoniae)

UDP-Glucose

NmLgtB (N. meningitidis)

UDP-Galactose

ApNGT (A. pleuropneumoniae)

UDP-Glucose

NmLgtB (N. meningitidis)

UDP-Galactose

LgtA (N. meningitidis?)

UDP-GlcNAc

ApNGT (A. pleuropneumoniae)

UDP-Glucose

NmLgtB (N. meningitidis)

UDP-Galactose

Glc/Gal

Glc/Gal/ GlcNAc

Glc/Gal/Sia (α2-6)

PdST6 (P. damsale)or
ST6Gal1 (H. sapiens)
ApNGT (A. pleuropneumoniae)
Glc/Gal/Sia (α2-3)

CMP-Sialic acid
UDP-Glucose

NmLgtB (N. meningitidis)

UDP-Galactose

CjCST-I (C. jejuni)

CMP-Sialic acidin vitro Glycosylation (IVG) of target proteins combinedwith Cell-Free Protein synthesis (CFPS) by PUREfrex®

Cell-free Protein Synthesis (CFPS)in vitro Glycosylation (IVG)
The glycosylation reaction was based on the method of Jewett et al.
(Kinghtlinger et al. (2019) Nat. Commun., 10, 5404).

Glycosylated Protein

GlycosylatedProtein

in vitro Glycosylation of model protein (IVG)
Colicin-E7 immunity protein mutant (Im7-6) (E. coli)
14693

ATT

GGNWTT AGG

LengthMolecular weight

6x His

101 a.a.
11,366 Da

glycosylation recognition sequence by ApNGT

lm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK(kDa)

lane
No.

1234

UDPUDPUDP-

Number of PSMs*Modification

Theo. MH+[Da]lane 1lane 2lane 3lane 4-3129.5739

50

18215Glc

3291.6267

0337910

Glc-Gal3453.6795

10648

Glc-Gal-(GlcNAc-Gal)13818.8117

029

Glc-Gal-(GlcNAc-Gal)24183.9439

049

Glc-Gal-(GlcNAc-Gal)34549.0761

030

20MS
analysis/ ApNGT/ NmLgtB/ LgtA

Im7-6Glucose
Galactose
GlcNAc*Number of PSMs (Peptide Spectrum Matches) was calculated as the average valueof three measurements.
(The same applies to the following experiments.)

IVG with PUREfrex® could provide the N-glycosylation of Im7-6.

in vitro Glycosylation of model protein (IVG)
Colicin-E7 immunity protein mutant (Im7-6) (E. coli)
146

93

ATT

GGNWTT AGG

LengthMolecular weight

6x His

101 a.a.
11,366 Da

glycosylation recognition sequence by ApNGT(kDa)

lane
No.

1

23

lm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK4MS
analysis

15

Theo. MH+[Da](control)lane 2lane 3lane 4-3129.5739

92754Glc

3291.6267

0302530

Glc-Gal3453.6795

03674

69

Glc-Gal-Sia

3744.7750

0777472

10

UDPUDPCMPCMPCMP-/ ApNGT/ NmLgtB/ PdST6/ ST6Gal1/ CjCST-I

Im7-6
Glucose
Galactose
Sialic acid

Number of PSMs

Modification

IVG with PUREfrex® could provide the addition of sialic acid to Im7-6.

One-pot IVG
IVG

One-pot IVG

Target Protein DNA

GTsDNACFPS
Sugar donor

CFPS
IVG
Glycosylated Protein

Glycosylated Protein

IVG

One-pot IVG
Colicin-E7 immunity protein mutant (Im7-6) (E. coli)

(kDa)

lane
No.

1234lm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK

20MS
analysis

1510

UDPUDPUDP-/ ApNGT/ NmLgtB/ LgtA

Im7-6Glucose
Galactose
GlcNAcNumber of PSMs

Modification

Theo. MH+[Da]

lane 1lane 2lane 3lane 4-3129.5739

9075070Glc

3291.6267

1*114

3121

Glc-Gal3453.6795

1*010251

Glc-Gal-(GlcNAc-Gal)13818.8117

00071

Glc-Gal-(GlcNAc-Gal)24183.9439

00034

Glc-Gal-(GlcNAc-Gal)34549.0761

0008*It is most likely detecting a carryover from the previous measurement.

One-pot IVG by PUREfrex® could provide the N-glycosylation of Im7-6.

in vitro Glycosylation of model protein (IVG)
Human Interleukin-6 (hIL6)
disulfide bond
73

30C78

ATT

GGNWTT

AGG C

212

FLAG

6x His72(Met)glycosylation recognition sequence by ApNGT
7278

Original sequence

CNKSNMC

Modified sequence

CATTGGNWTTAGGC

Length209 a.a.

Molecular weight

23,550 Da

PDBj: 2IL6

in vitro Glycosylation of model protein (IVG)
Human Interleukin-6 (hIL6)
disulfide bond
7330C78

ATT

GGNWTT

AGG C

212

FLAG

6x His72(Met)glycosylation recognition sequence by ApNGT
7278

CNKSNMC

Original sequence(kDa)25

CATTGGNWTTAGGC

Modified sequenceLength

209 a.a.

Molecular weight

23,550 Da

20

UDPUDPUDPCMPCMP-/ ApNGT/ NmLgtB/ LgtA/ ST6Gal1/ CjCST-I

IVG by PUREfrex® could not provide the N-glycosylation of Im7-6.

hIL-6
Glucose
Galactose
GlcNAcSialic acid

Multi-step IVG

CFPS
Targetprotein

GT 1GT 2GT 3+
Targetprotein+GT 1+Sugar
donor+GT 2+Sugardonor+GT 3Sugar
donor

IVG-1

IVG-2

IVG-3

Ni-resinpurification

Ni-resinpurification

Ni-resinpurification

Multi-step IVG

Multi-step IVG
Human Interleukin-6 (hIL6)

Human Granulocyte-macrophage colonystimulating factor (hGM-CSF)

(kDa)
(kDa)

20

25
2015

UDPUDPUDPCMPCMP-/ ApNGT/ NmLgtB/ LgtA/ ST6Gal1/ CjCST-I

hIL-6
Glucose
Galactose
GlcNAcSialic acid

UDPUDPUDPCMPCMP-/ ApNGT/ NmLgtB/ LgtA/ ST6Gal1/ CjCST-I

hGM-CSF
Glucose
Galactose
GlcNAcSialic acid

Multi-step IVG with PUREfrex® was effective for disulfide-bonded proteins.

Multi-step IVG
Human Beta-2 adrenergic receptor (hADRB2)
cytoplasmic domain

extracellular domain
815ATT(Met)GGNWTT

AGG
transmembrane

Length416 a.a.

Molecular weight

46,764 Da

glycosylation recognition sequence by ApNGT
1520

Original sequence

NGSHAP

Modified sequence

ATTGGNWTTAGGHAP

CFPSMulti-step
IVG

Phospholipid bilayer

Membrane scaffold
protein (MSP)

Nanodisc
Incubation

DNA encoding Membrane Protein

Membrane Protein
in Nanodisc

Glycosylated Membrane Protein

Multi-step IVG
Human Beta-2 adrenergic receptor (hADRB2)

(kDa)
50

hADRB2/MSP1E3D1-DMPG
Glucose
Galactose

37(MSP1E3D1-His6)
25

UDPUDP-Multi-step
IVG

Membrane Protein
in Nanodisc

Glycosylated
Membrane Protein/ ApNGT/ NmLgtB

Multi-step IVG with PUREfrex® was effective formembrane proteins.

Summary• N-glycosylation of Im7-6 were succeeded IVG and One-pot IVG withPUREfrex®.
• Depending on the target protein, glycosylation could be achieved byarranging the method as Multi-step IVG.

Glycoproteins can be synthesized by IVG using PUREfrex®.