®
PUREfrex を用いた

ジスルフィド結合含有タンパク質の合成
ジーンフロンティア株式会社
金森崇2017年12月6日 ConBio2017

Topics®
1. Introduction of PUREfrex®
2. Application of PUREfrex•Optimization of nt sequence at 5’-terminus of ORF•Synthesis of proteins containing disulfide bonds•Synthesis of antibody-related proteins

PURE system
Components of the original PURE system

Outline of protein synthesis in E. coli

Purified Transcription/Translation Factors
T7 RNA Polymerase
Initiation Factors (IF1, IF2, IF3)
Elongation Factors (EF-Tu, EF-Ts, EF-G)
Release Factors (RF1, RF2, RF3)
Ribosome Recycling Factor
20 Aminoacyl-tRNA synthetases (ARS)
Methionyl-tRNA transformylase (MTF)
E. coli Ribosome
E. coli tRNA mix
Energy Regeneration System(Enzymes and Substrates)
4 NTPs20 Amino Acids
K+, Mg2+
Polyamine
Reductant
BufferShimizu et al. (2001) Nat. Biotechnol., vol.19, p.751
Shimizu et al. (2005) Methods, vol.36, p.299

Development of PUREfrex®
Summary of the preparation methods of the components
Original
PURE system

PUREfrex®

His-Tag

None

Number of columns

1>3

Wash with detergent-+-+-+ProteinTagRibosome
Wash with detergenttRNAWash with detergent

Original PURE system: Shimizu et al. (2005) Methods, vol.36, p.299

Comparison of E. coli-based cell-free protein synthesis system
Extract System

Reconstituted System

S30 system

Original
PURE system

PUREfrex®

100-1000 µg/mL

10-200 µg/mL

10-200 µg/mL

RNase

very HighLow

very Low

LPSvery HighHighvery Low

Plasmid DNA

OKOK

OK

PCR product

NG

OK

OK

Customization ofReagent

Difficult

Easy

Easy

Purification of Histagged product

OKNG

OK

Typical Yield
Contamination

Template DNA

Original PURE system: Shimizu et al. (2005) Methods, vol.36, p.299Comparison of E. coli-based cell-free protein synthesis system
Extract System

Reconstituted System

S30 system

Original
PURE system

PUREfrex® 2.0

100-1000 µg/mL

10-200 µg/mL

20-1000 µg/mL

RNase

very HighLow

very Low

LPSvery HighHighvery Low

Plasmid DNA

OKOK

OK

PCR product

NG

OK

OK

Customization ofReagent

Difficult

Easy

Easy

Purification of Histagged product

OKNG

OK

Typical Yield
Contamination

Template DNA

Original PURE system: Shimizu et al. (2005) Methods, vol.36, p.299

Protein synthesis using PUREfrex® 1.0 and 2.0A) SDS-PAGE

B) Fluorescence of synthesized GFPTopics®
1. Introduction of PUREfrex®
2. Application of PUREfrex•Optimization of nt sequence at 5’-terminus of ORF•Synthesis of proteins containing disulfide bonds•Synthesis of antibody-related proteinsEffect of 5’-terminal nucleotide sequence on the yield
Trastuzumab Heavy chain (VH+CH1)

Effect of 5’-terminal nucleotide sequence on the yield
Trastuzumab Heavy chain (VH+CH1)

High

Tested clones : 56 clones

Synthesized amountLow

Effect of 5’-terminal nucleotide sequence on the yield
Trastuzumab Heavy chain (VH+CH1)

Tested clones : 56 clones

Effect of 5’-terminal nucleotide sequence on the yield
Trastuzumab Heavy chain (VH+CH1)

Tested clones : 56 clones

Effect of 5’-terminal nucleotide sequence on the yield
Trastuzumab Heavy chain (VH+CH1)

Tested clones : 56 clones

AT-rich codon > Major codon@ 5’-terminus of ORF

Topics®
1. Introduction of PUREfrex®
2. Application of PUREfrex•Optimization of nt sequence at 5’-terminus of ORF•Synthesis of proteins containing disulfide bonds•Synthesis of antibody-related proteins

Synthesis of functional proteins using PUREfrex®

Translation process
Synthesis ofpolypeptide chain

Polypeptide

Synthesis of functional proteins using PUREfrex®
Polypeptide ≠ Functional protein
Translation process
Synthesis ofpolypeptide chain

Maturation process•Formation of higher-orderstructure• Folding of polypeptide• Formation of complex•Post-translational modification• Formation of disulfide bonds• Phosphorylation• Glycosylation• Lipidation• Processing•Localization• Insertion into membrane• Translocation across membrane

Common process

Diversified processfunctionalprotein

Synthesis of functional proteins containing disulfide bonds
Polypeptide ≠ Functional protein
Translation process
Synthesis ofpolypeptide chain

Maturation process•Formation of higher-orderstructure• Folding of polypeptide• Formation of complex•Post-translational modification• Formation of disulfide bonds• Phosphorylation

functionalprotein• Glycosylation• Lipidation• Processing•Localization• Insertion into membrane• Translocation across membrane

Common process

Diversified processoxidative condition

Synthesis of functional proteins containing disulfide bonds
Reductant
Dithiothreitol (DTT)

Tris(2-carboxyethyl)phosphine (TCEP)

Glutathione (GSH)

Cysteine (Cys)

3-Mercapto-1,2-propanediol (MPD)

2-Mercaptoethanol (2-ME)

Synthesis of functional proteins containing disulfide bonds
E. coli alkaline phosphatase (AP) (2 disulfide bonds)

Synthesis of functional proteins containing disulfide bonds
E. coli acid phosphatase (AppA) (5 disulfide bonds)+ DsbC (E. coli disulfide isomerase)

New productTopics®
1. Introduction of PUREfrex®
2. Application of PUREfrex•Optimization of nt sequence at 5’-terminus of ORF•Synthesis of proteins containing disulfide bonds•Synthesis of antibody-related proteins

Synthesis of scFv and FabTrastuzumab-derived scFv and FabA) SDS-PAGEB) ELISA

Yield: 0.4 mg/mL

0.5 mg/mL

Synthesis of aglycosylated IgG
Trastuzumab-derived aglycosylated IgG

Key Factors•HC/LC DNA ratio•Reductant•Molecular chaperones•Incubation Temperature•Incubation Time

Synthesis of aglycosylated IgG
Trastuzumab-derived aglycosylated IgG

Key Factors•HC/LC DNA ratio•Reductant•Molecular chaperones•Incubation Temperature•Incubation Time

Synthesis of aglycosylated IgG
Trastuzumab-derived aglycosylated IgGA) SDS-PAGE

B) ELISA

Yield:0.12 mg/mL

Product List

タンパク質合成反応液
New

添加剤
DnaK/DnaJ/GrpE
GroEL/GroES
Oxidized Glutathione, DsbC