再構成型無細胞タンパク質合成系 (PUREfrex®) および糖転移酵素を用いた糖タンパク質合成の試み
Synthesis of glycosylated proteins using PUREfrex® with glycosyltransferase○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)○ Rena Matsumoto, Tatsuya Niwa, Hideki Taguchi and Takashi Kanamori (1GeneFrontier Corp., 2Cell Biology Center, IIR, Tokyo Tech)<Abstract>

in vitro Glycosylation (IVG) of target proteins combined with Cell-Free Protein
synthesis (CFPS) by PUREfrex®

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。そのため、反応条件や追加因子によって、合成したタンパク質の翻訳後修飾の調節が容易である。これまでに酸化剤とプロテインジスルフィドイソメラーゼ (PDI) を追加して活性を有したジスルフィド結合含有タンパク質の合成などが可能であることを示してきた。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てN型糖鎖修飾タンパク質をPUREfrex®で合成することが可能かを検討した。
糖鎖修飾反応はJewettらの方法を参考にした (Kinghtlinger et al. (2019) Nat. Commun., 10, 5404)。大腸菌
Colicin-E7 immunity protein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。IVG後のSDS-PAGEおよび質量分析の結果、Im7-6の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、酵素依存的にガラクトース、GlcNAcあるいはシアル酸の付加を確認した。さらに、
Im7-6と糖鎖転移酵素の合成および糖鎖修飾反応を同時に行うone-pot合成・糖鎖修飾反応(cell-freeproteinsynthesis (CFPS)-IVG one-pot)を行ったところ、この方法でも糖鎖修飾されたIm7-6を確認できた。
また、ジスルフィド結合タンパク質や膜タンパク質についても、IVGの方法を変えることによって、糖鎖付加が可能であることが分かった。
以上の結果より、 PUREfrex®でも糖タンパク質を合成できることが示された。

Reconstituted cell-free protein synthesis system

Cell-free Protein Synthesis (CFPS)

based on the PURE system technology

AdvantageApplication

・Low level of contaminationin vitro Glycosylation (IVG)

・High throughput preparation of proteins

・Easy adjustment of theThe glycosylation reaction was based on the method of Jewett et al.

(including Fab, scFv, protein toxin etc.)
・Protein science research

reagent composition
・PCR products usable as a

(Kinghtlinger et al. (2019) Nat. Commun., 10, 5404).

・in vitro display

Glycosylated Protein

template DNA(ribosome display, mRNA display etc.)(Ref: Shimizu et al. (2001) Nat. Biotechnol., vol. 19, p. 751.)

1. in vitro Glycosylation of model protein
1-1. IVG

1-2. One-pot IVG
Colicin-E7 immunity protein mutant (Im7-6) (Escherichia coli)

Cell-free Protein synthesis (CFPS) of target protein andGlycosyltransferases (GTs)14693ATTGGNWTT AGG

LengthMolecular weight

6x HisTarget ProteinDNA

101 a.a.
11,366 Da

CFPS

GTsDNA

IVG

Sugar donor

glycosylation recognition sequence by ApNGT

One-pot CFPS-IVG condition
lm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK(kDa)

Number of PSMs*

Modification

Theo. MH+[Da]

lane 1lane 2lane 3lane 4-3129.5739

50

182

15

Glc

3291.6267

033

7910

Glc-Gal3453.6795

10648

Glc-Gal-(GlcNAc-Gal)13818.8117

029

Glc-Gal-(GlcNAc-Gal)24183.9439

049(kDa)

Glc-Gal-(GlcNAc-Gal)34549.0761

0302020

UDPUDPUDP-MSanalysis/ ApNGT

Im7-6
Glucose
Galactose
GlcNAc/ NmLgtB/ LgtA

Template DNA

Reaction mixture

Sugar donor

Im7-6 (3.7 nM)
ApNGT (0.2 nM)
NmLgtB (0.1 nM)
LgtA (0.5 nM)

PUREfrex® 2.1

UDP-glucose (2.5 mM)
UDP-galactose (2.5 mM)
UDP-GlcNAc (2.5 mM)

Glycosylated Protein

One-pot IVG by PUREfrex®

*Number of PSMs (Peptide Spectrum Matches) was calculated as the average value ofthree measurements.
(The same applies to the following experiments.)

15

could provide the Nglycosylation of Im7-6.

10

UDPUDPUDP-/ ApNGT

Im7-6Glucose
Galactose
GlcNAc/ NmLgtB/ LgtA

lm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK

GlycosylatedProtein

MSanalysis(kDa)
15→ All IVG samples were purified using Ni-Sepharose 6 FF.

UDPUDPCMPCMPCMP-→ Eluates were applied to SDS-PAGE(10-20%).
Gel staining: Oriole Fluorescent Gel Stain (Bio-Rad)
Quantitation: LAS-4000 system (GE Healthcare)/ ApNGT/ NmLgtB/ PdST6/ ST6Gal1/ CjCST-I

Im7-6
Glucose
Galactose
Sialic acidlm7-6 [21-49] EIEKEATTGGNWTTAGGDVLDVLLEHFVK

Number of PSMs

Modification

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

92754

Glc

3291.6267

030

2530

Glc-Gal3453.6795

03674

69

Glc-Gal-Sia

3744.7750

07774

72

10MSanalysis→ The rest of the samples were subjected to mass spectrometry.
(Q-Exactive & Proteome Discoverer 2.4)

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

2. in vitro Glycosylation of disulfide bonded protein+Targetprotein

Targetprotein

GT 1(4 mM GSH)
+ DnaK Mix

GT 2

Theo. MH+[Da]

lane 1lane 2lane 3lane 4-3129.5739

9075070

Glc3291.6267

1*114

3121

Glc-Gal3453.6795

1*010251

Glc-Gal-(GlcNAc-Gal)13818.8117

00071

Glc-Gal-(GlcNAc-Gal)2

4183.9439

00034

Glc-Gal-(GlcNAc-Gal)34549.0761

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

3. in vitro Glycosylation of membrane protein+GT 1

Number of PSMs

Modification++Sugar
donor+Sugar
donor

GT 2+Sugar
donor

GT 3

IVG-1

IVG-2

IVG-3

Ni-resinpurification

Ni-resinpurification

Ni-resinpurification

CFPS
Multi-step
IVG

Phospholipidbilayer
Membrane
scaffoldprotein (MSP)

Nanodisc

GT 3Incubation

DNA encoding Membrane Protein

CFPS

Membrane Protein
in Nanodisc

Glycosylated
Membrane Protein

Multi-step IVG

Human Beta-2 adrenergic receptor (hADRB2)
Human Interleukin-6 (hIL6)

Human Granulocyte-macrophage colony-stimulating factor(hGM-CSF)

733078C ATT

GGNWTT AGG C

FLAG

6x His184447

ATT

glycosylation recognition sequence by ApNGT
7278

Length209 a.a.
Molecular weight 23,550 Da

GGNWTT AGG

113CC CC

105

138

434449(Met)144

disulfide bond

NLSRDT

Original sequence
Modified sequence

71

(Met)
glycosylation recognition sequence by ApNGTCNKSNMC

Original sequence

ATT

212

72

(Met)

15

disulfide bonddisulfide bond

FLAG

Length416 a.a.
Molecular weight 46,764 Da

GGNWTT AGG
transmembrane

glycosylation recognition sequence by ApNGT

6x HisLength155 a.a.
Molecular weight 17,475 Da

cytoplasmic domain

extracellular domain
81520

Original sequence

NGSHAP

Modified sequence

ATTGGNWTTAGGHAP(kDa)

CATTGGNWTTAGGC
Modified sequence

ATTGGNWTTAGGRDT50hADRB2/MSP1E3D1-DMPG
Glucose
Galactose

37

IVG

IVG
(MPS1E3D1-His6)
(kDa)

(kDa)

25

2025?

20

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

hIL-6
Glucose
Galactose
GlcNAcSialic acid

UDPUDP-/ ApNGT/ NmLgtB

Glucose and Galactose were added to asparagine

15

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

hGM-CSFGlucose
Galactose
GlcNAcSialic acidin hADRB2 by multi-step IVG with PUREfrex®.

<Summary>
Multi-stepMulti-step

IVG

IVG
(kDa)(kDa)

202520

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

hIL-6
Glucose
Galactose
GlcNAcSialic acid15

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

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

Future Study• Application of current glycosylation methods to other proteins.
• Examination of oligosaccharide glycosylation by IVG with PUREfrex® .

hGM-CSF
Glucose
Galactose
GlcNAcSialic acid• N-glycosylation of Im7-6 were succeeded IVG and One-pot IVG
with PUREfrex®.
• Depending on the target protein, glycosylation could beachieved by arranging the method as Multi-step IVG.

Glycoproteins can be synthesized by IVG using
PUREfrex®.
For more information, please contact us.
URL: www.genefrontier.com
E-mail: purefrex@genefrontier.com