advanced, flexible, proven, patented* cell-free translation translation and energy recycling. In...

PURESYSTEM®PURESYSTEM®In Vitro Protein Synthsis KitIn Vitro Protein Synthsis Kit

advanced, flexible, proven, patented*cell-free translation

*Patent NumberUS : 7,118,883EU : EP131907B1

• PURESYSTEM kits pre-configured for producing

unmodified proteins

folded proteins

S-S bonded proteins

or

• PURESYSTEM ∆ kits for translation research.

• Translated proteins are tag free.

• Highly defined recombinant HIS-tagged translation components for easy protein

purification and precision reaction controllability.

• Simple three hour protocol.

www.cosmobio.co.jp

Contents

Optional Products

Custom

PURESYSTEM Technology 2

PURESYSTEM® Kit 3

PURESYSTEM® classic II 4

PURESYSTEM® S-S 5

PURESYSTEM® Duo 6

pURE Vector 8

AP template & substrate 6

Universal Primer 8

PURESYSTEM® custom 9

Priduct List

PURESYSTEM® ∆series 10

PURESYSTEM® Kit/Optional Products 11

1

PURESYSTEM® Advance 7

PURESYSTEM Technology

What's "PURESYSTEM Technology"?What's "PURESYSTEM Technology"?

Ribosome

His-Tagged Transcription/Translation Factors

tRNA, NTPsAmino AcidsSalts, Buffer

Basic Components of PURESYSTEM TechnologyHis-Tagged 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 Methionyl tRNA transfolmyrase

E. coli RibosomeE. coli tRNAEnergy Regeneration SystemNTPs, Amino Acids, Salts, Buffer

PURESYSTEM is a novel reconstituted in vitro translation system which consists of about 30 purified enzymes necessary for transcription, translation and energy recycling.*

In PURESYSTEM, all the factors for transcription and translation are tagged with a hexahistidine; 3 initiation factors (IF1, IF2, IF3), 3 elongation factors (EF-G, EF-Tu, EF-Ts), 3 release factors (RF1, RF2, RF3), ribosome recycling factor, 20 aminoacyl-tRNA synthetases, methionyl-tRNA formyltransferase and T7 RNA polymerase.

The reagents also contains E. coli 70S ribosome, amino acids, NTPs, E.coli tRNA and energy recycling system, so the target protein can be synthesized just by addition of the template DNA to the reaction.

* Shimizu Y., et al., (2001) Nature Biotechnology, vol.19, p.751-755.

PURESYSTEM Technology has unique characters because of this is an only composition technology in the world.

Pure Reaction MixPure Reaction Mix

No Tags RequiredNo Tags Required

Modification AvailableModification Available

Time SavingTime SavingAll you need is to follow the 3 steps as shown in next page. It takes

only 10 minutes for your handling and 3 hours for incubation and

centrifugation.

Because PURESYSTEM is a reconstituted system in which all the

components are identified, the reagents in PURESYSTEM classic II

are essentially free from unidentified components. Therefore, the

synthesized product is hardly susceptible to unwanted degradation

or modification.

All the proteinous factors are His-tagged and no unknown

components are present within the reaction, the synthesized

protein can be easily purified by removal of the His-tagged factors

and ribosome using metal affinity resin and ultrafiltration

membrane, respectively. Therefore, no unexpected effects of tags

needs to be considered (e.g.; misfolding or aggregation), promising

PURESYSTEM is a reconstituted system, so the reagent composition

can be easily modified according to the purpose of experiment. This

can be ordered as PURESYSTEM custom, which is made upon

request.

2

Protein expression

1 2 3 4 5 6 7 8 9

14.4

20

30

45

66

97.4kDa

• Analysis of the protein interaction

• Epitopic mapping

• The cyclopedic composition / functional analysis

of the DNA library

• Various mark proteosynthesis

• A study of the protein folding

PURESYSTEM® Kit

PURESYSTEM®Template DNA

Ribosome

His-Tagged Factors

Metal Affinity Resin

Synthesized Protein

Addition of template DNA

starts protein synthsis reaction.

Addition of metal affinity resin

for adsorption of tagged factors.

Synthesized Protein

can be isolated.

A B

or

Schematic diagram of protein synthesis and purification by PURESYSTEM®

Removal of Ribosome and resin

with tagged factors by

Ultrafiltration.

β-ga

lact

osid

ase

DHFR

DnaJ

Enol

ase

CAT

MAP

kin

ase

Ras

MW

mar

ker

IL8

Applications

PURESYSTEM® Kit fits both PCR product and Plasmid as template DNA. It is essential that templates have

• Start Codon (ATG)

• Stop Codon(TAG or TGA or TAA)

• T7 Promoter during initiation.

• Ribosome binding site lies about 10 nucleotides upstream from the start codon.

• In the case of PCR products, 6 nucleotides downstream from the stop codon.

( Transcription terminator is no necessary .)

• In the case of plasmid DNA, transcription terminator downstream from the stop

codon.

Materials for using PURESYSTEM® Kit

Template DNA

PURESYSTEM® Kit is based on E.coli translation makes the

production of prokaryotic enzymes. And a few eukaryotic enzymes

were made and shown to be functional.

PURESYSTEM® Kit can compose protein of up to 100 µg per

1mL.

Water Bath

Metal affinity chromatography , Ultrafiltration

PURESYSTEM® Kit is not contain elements for purification of

synthesized protein. Please prepare metal affinity chromatography and

ultrafiltration when you use the protocol above. About the use of these

products, please refer to the instruction manual of each product.

Molecular biology experiment, an apparatus

Expression and reverse purification1 2 3 4 5

DHFR

9766

45

30

20.1

14.1

1. Marker 2. No DNA3. total protein4. Ultrafiltration flow through

(Removal of Ribosome)5. Ni-NTA Agarose flow through (Removal of His-Tagged Factors)

kDa

3

PURESYSTEM® classic II


PURESYSTEM® classic II standard

PURESYSTEM® classic II mini

PURE2048C

PURE2030C

PURE2004C

Cat.# Product Name Product size Price

50µl x 96 reactions

50µl x 8 reactions

1ml x 3 reactions

4

Contents

Temp.DHFR

Univ.Primer

Sol.A

Sol.B

5µl x 1

80µl x 1

25µl x 8

10µl x 8

mini

50µl x 1

80µl x 1

500µl x 3

200µl x 3

standard

5µl x 2

500µl x 2

25µl x 96

10µl x 96

96

PURESYSTEM is a novel reconstituted in vitro translation system which

consists of about 30 purified enzymes necessary for transcription,

translation and energy recycling. In PURESYSTEM, all the factors for

transcription and translation are tagged with a hexahistidine. The reagents

also contains E. coli 70S ribosome, amino acids, NTPs, E.coli tRNA and energy

recycling system, so the target protein can be synthesized just by addition

of the template DNA to the reaction.

Most of the inhibitors, RNase and protease, of the proteosynthesis are not

included in this reagent.

Store at -80ºC Avoid multiple freeze-thaw cycles

classic IIclassic

Purity Mixture of RNase in about 1/10

Quantity of composition More than doubl

Stop Codon Useage non-response by UGA cope with all of

UAG/UGA/UAA

Tagged with components A part of the energy

regenerating system enzyme is

non-addition.

add it to all factors except

the ribosomal protein.

PURESYSTEM classic II Features

Q

uan

tity

(rel

ativ

e va

lue)

1

2

0classic

Quantity of Composition

1

200

0classicKit A

RN

ase

acti

vity

(rel

ativ

e va

lue)RNase activity

classic II

classic II

PURESYSTEM® S-S

PURESYSTEM® S-S 96

PURESYSTEM® S-S standard

PURESYSTEM® S-S mini

PURE3048S

PURE3030S

PURE3004S

50µl x 96 reaction

50µl x 8 reaction

1ml x 3 reaction

5

Contents

Sol.A

Sol.B

Univ.Primer

25µl x 825µl x 96

10µl x 810µl x 96

80µl x 180µl x 1500µl x 2

500µl x 3

200µl x 3

ministandard96

PURESYSTEM S-S is especially designed to enable the formation of

disulfide bond(s) of synthesized protein. This was previously believed to be

impossible with conventional in vitro protein synthesis systems including E.

coli S30 extract, wheat germ extract and rabbit reticulocyte lysate systems.

The high purity and flexibility of PURESYSTEM enables the redox

conditions of reaction mixture to be reproducible. PURESYSTEM S-S

consists solely of defined components and does not contain any

contaminants such as glutathione reductase and thioredoxin reductase

which may cause redox reactions.

In PURESYSTEM S-S, you only add template DNA of the purpose protein

to reaction liquid and can compose protein having disulfide combination.

The additional processing after other additives and synthesis reactions is

not necessary.

* These kits do not include template DHFR .

Composition/enzyme activity of Alkaline phosphatase

Only PURESYSTEM® S-S composed a certain active Alkaline phosphatase. (ND: Not Determined)

classic II

ND

100

50

0

100

50

0

com

po

siti

on

(%)

enzy

me

acti

vity

(%) enzyme activity

composition


competitorA

S-S


PURESYSTEM® Duo

6

PURESYSTEM Duo is an introductory in vitro transcription/translation

kit which contains both PURESYSTEM classic II (reduced condition)

and PURESYSTEM S-S (oxidized condition) reagents. PURESYSTEM

S-S is especially designed to enable the formation of disulfide bond(s) of

synthesized protein. This was previously believed to be impossible with

conventional in vitro protein synthesis systems including E. coli S30 extract,

wheat germ extract and rabbit reticulocyte lysate systems. The high purity

and flexibility of PURESYSTEM enables the redox conditions of reaction

mixture to be reproducible.

Contents

Sol.A Univ.Primer25µl x 4 80µl x 1S-S

classic IISol.B 10µl x 4 Temp.DHFR 5µl x 1

Sol.A 25µl x 4 Temp.AP 15µl x 1

Sol.B 10µl x 4 Sub.AP 1mg x 1

Buf.AP 1ml x 1

AP template & substrate

AP template & substratePUREAP01

Temp.AP 15µl x 1

Sub.AP 1mg x 1

Buf.AP 1ml x 1

DNA encoding AP (Alkaline Phosphatase) and a substrate and a buffer for

enzyme activity measurement of complex AP are set products.

You can use it as control when you use PURESYSTEM® S-S.

PURESYSTEM® DuoPURE4004Dclassic II

S-S 50µl x 4 reaction50µl x 4 reaction


Store at -20ºC


Composition/enzyme activity of Alkaline phosphatase

Only PURESYSTEM® S-S composed a certain active Alkaline phosphatase. (ND: Not Determined)

classic II

ND

100

50

0

100

50

0

com

po

siti

on

(%)

enzy

me

acti

vity

(%) enzyme activity

composition

competitorA

S-S


PURESYSTEM® advance

PURESYSTEM® advance 96PURE5048A 50µl x 96 reactions

PURESYSTEM® advance standardPURE5030A 1ml x 3 reactions

PURESYSTEM® advance crystalPURE5100A 10ml x 1 reactions

7

Using PURESYSTEM advance, milligram scale protein is in your

hand within an hour and even purified protein is also in your hand

in next two hours. Since the experimental protocol is nearly same

as our PURESYSTEM classic II, users who arefamiliar with

classic II can start using an advance kit without new training. The

efficiency of protein synthesis of PURESYSTEM advance is more

than twice as much as that of classic II. In almost all case, the cost

of synthesis per protein amount is lower than that of classic II.

Quantity up synthetic with most protein

We composed DHFR, DnaJ, enolase, CAT, MAPkinase,

IL -8 and Ras in classic II and advance, and

compared the synthetic quantity by fluorescence

Labeling Law. Each lane 1 shows a complex case

using classic II. Lane 2 shows a complex case

using advance. We understood that we increased

when we compared the quantity of synthetic

protein (green in a figure) performed fluorescence

labelling of when we used classic II and used

advance.

Lane 1 : Using classic II

Lane 2 : Using advance

Synthetic in high efficiency of an average of

approximately 2 times

Showed quantity of relative proteosynthesis (when

I assumed our classic II 100%). Composition

efficiency improved it to approximately 185% on

258%, average with the thing which the most

synthetic efficiency improved.


eno

lase

eno

lase

Qu

anti

ty o

f co

mp

osi

tio

n (a

rela

tive

rati

o) A comparison between classic II and advance

Lane

8

pURE Vector

It is the vector which is most suitable for proteosynthesis using

PURESYSTEM®. There are four kinds of vectors that the restriction enzyme

parts of the cloning site are different. The most suitable vector has you

choose him by genetic sequence to introduce.

pURE1PUREV001

PUREV002

PUREV003

PUREV004

pURE2

pURE3

pURE4

10µg x 1

Others-Universal Primer/Template DHFR

Universal PrimerPURER001 500µl (2µM) x 1

DHFR plasmid DNAPUREDH001 100µl x 1

In the case of the second phase PCR, you use Universal Primer with a primer

for the PCR for template DNA preparations to use for proteosynthesis using

PURESYSTEM®. It includes T7 promoter and ribosome-binding sequence.

It is a product same as Univ.Primer included in PURESYSTEM®.

5'-GAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCA-3'

T7 promoter RBS (SD sequence)

Base sequence of Universal Primer

Store at -20ºC

Store at -20ºC

NdeIBamHISmaIEcoRIXhoIHindIII

NcoIBamHISmaIEcoRIXhoIHindIII

SphIBamHISmaIEcoRIXhoIHindIII

EcoRVBamHISmaIEcoRIXhoIHindIII

Amp

r

ori 2215 bp

T7 promoter

T7 terminator

MCS

pURE1pURE1 pURE2pURE2 pURE3pURE3 pURE4pURE4

5'-TAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTA

ACTTTAAGAAGGAGATATCATATGGCATCGGCGGATCCCGGGAATTCTCGAGAAGCTT

TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTT-3'

T7 promoter transcription

RBS

NdeI BamHI

SmaI XhoI

EcoRI HindIII

translationstart codon

ACCATGGNcoI

AGCATGCSphI

GATATCEcoRV

pURE2

pURE3

pURE4

pURE1

T7 terminator



PURESYSTEM® custom

PURESYSTEM® custom is only custom tailoring proteosynthesis

reaction liquid in the world born from structurelessness proteosynthesis

technology ... which reconstituted the factor which maximum good point -

of PURESYSTEM Technology is necessary for.

PURESYSTEM® custom prepares reaction liquid to the demand of the

visitor and contributes it. For example, I can offer following reaction liquid.

• Reaction liquid except the specific amino acid

• Reaction liquid in coordination with tRNA density

• Reaction liquid except the specific Buffer ingredient

• Reaction liquid except the ribosome

• Reaction liquid in coordination with ribosome density

• Reaction liquid except the specific protein factor

• The reaction liquid that regulated the density of

specific protein factor

For information about PURESYSTEM technology and kits

[email protected]

9

T7 RNA polymeraseT7 RNA polymerase

IF1IF1

IF2IF2

IF3IF3

RF1RF1

RF2RF2

RF3

EF-TuEF-GEF-G

EF-TsEF-Ts

ARSsARSs

MTFMTF

RibosomeRibosome

tRNAtRNA

NTPsNTPs

AAsAAs

SaltsSalts

BufferBuffer arrangement of AA concentration

Ribosome only

arrangement of tRNA concentration

no addition of RF1

RRFRRF

PURESYSTEM® custom is made-to-order PURESYSTEM®

custom reaction liquid to perform build-to-order manufacturing

to the demand of the customer. In PURESYSTEM® custom, we

may usually disclose the information that we do not disclose.

Therefore, in the case of the purchase, we may have you conclude

the contract including the secret maintenance article by all

means.

All intellectual property rights to affect PURESYSTEM®

custom belong to us. Therefore a result using PURESYSTEM®

custom comes to need the discussion with us about the

handling of the improved invention that occurred. But the object

needing discussion becomes only improved invention of

PURESYSTEM® custom in itself and the allied result. Because I

may demand the disclosure of results of research in the case of

the discussion concerned as far as it is necessary, approve it

beforehand.

Please refer for any questions to us.

On the occasion of the purchase of PURESYSTEM® custom

PURESYSTEM®∆series

PURESYSTEM® ∆1 RF1PDS101



PURESYSTEM® ∆1 RRFPDS104

PURESYSTEM® ∆1 EF-GPDS105

PURESYSTEM® ∆1 tRNA PolymerasePDS107

PURESYSTEM® ∆1 RibosomePDS108

PURESYSTEM® ∆1 PheRSPDS109

PURESYSTEM® ∆1 IleRSPDS110

PURESYSTEM® ∆1 AlaRSPDS111

PURESYSTEM® ∆1 TrpRSPDS112

PURESYSTEM® ∆1 tRNAPDS113

PURESYSTEM® ∆1 ArgPDS114

PURESYSTEM® ∆1 MetPDS115

PURESYSTEM® ∆1 TrpPDS117

PURESYSTEM® ∆2 RF1,RF2PDS201

PURESYSTEM® ∆2 tRNA,RF2PDS202

PURESYSTEM® Suppli RF1PSPRF1



PURESYSTEM® Suppli RRFPSPRRF

PURESYSTEM® Suppli EF-GPSPEFG

PURESYSTEM® Suppli tRNA PolymerasePSPT7R

PURESYSTEM® Suppli PheRSPSPPRS

PURESYSTEM® Suppli IleRSPSPIRS

PURESYSTEM® Suppli AlaRSPSPARS

PURESYSTEM® Suppli TrpRSPSPTRS

PURESYSTEM® Suppli tRNAPSPTRN

PURESYSTEM® Suppli ArgPSPARG

PURESYSTEM® Suppli MetPSPMET

PURESYSTEM® Suppli TrpPSPTRP

PURESYSTEM® Suppli RibosomePSPR

10

50µl x 8 reaction

50µl x 8 reaction

Quantity equal toquantity of additionto PURESYSTEM®

reaction liquid400µL

same as the above


Reconstitute Protein Synthesis

PURESYSTEM® Kit

Others (Vector, Primer, Control for S-S)


PURESYSTEM® classic II standard

PURESYSTEM® classic II mini

PURESYSTEM® S-S 96

PURESYSTEM® S-S standard

PURESYSTEM® S-S mini

PURESYSTEM® Duo

pURE1

pURE2

pURE3

pURE4

Universal Primer

AP template & substrate

PURE2048C

PURE2030C

PURE2004C

PURE3048S

PURE3030S

PURE3004S

PURE4004D

PURER001

PUREV001

PUREV002

PUREV003

PUREV004

PUREAP01

50µl x 96 reaction

50µl x 96 reaction

50µl x 8 reaction

50µl x 8 reaction

1ml x 3 reaction

1ml x 3 reaction

classic IIS-S 50µl x 4 reaction

50µl x 4 reaction

Custom service

PURESYSTEM® custom

10µg

2µM x 500µl

DHFR plasmid DNAPUREDH001 100µL x 1

Temp.AP (15µl x 1)Sub.AP (1mg x 1)Buf.AP (1ml x 1)

We provide the reaction liquid that we prepared to the demand of the customer including the component / reaction liquid quantity.

*The specifications of PURESYSTEM® and the product concerned, a standard, the price may be changed without a notice.

[Manufacturer][Manufacturer]

BioComber CO.,LTD.BioComber CO.,LTD.

Reconstitute Protein Synthesis



[Distributor]

advanced, flexible, proven, patented* cell-free translation translation and energy recycling. In...

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