accelerating protein research

7/18/2013 Matthias Harbers 1

Dr. Matthias Harbers – Vice President Sales and Marketing Division

Accelerating Protein Research

CellFree Sciences

Reagents – Protein Synthesizers – Protein Expression Services

CellFree Sciences 7/2013


CellFree Sciences offers a cell-free protein expression platform

technology to rapidly deliver proteins for a broad range of

applications:

- ENDEXT® Technology -

What We Can Offer

Our Platform Technology Is Accelerating Protein Research!


The Company

CellFree Sciences Co., Ltd.

Based on ENDEXT® Technology

developed in the laboratory of

Prof. Y. Endo

Protein expression reagents

Protein synthesizers

Protein expression services

Contract research

Technology licensing

Yokohama Business Headquarters

Matsuyama Headquarters

( Ehime University)

Established July 2002 at Ehime University, Japan


Cell-free protein synthesis is the preferable protein preparation method for:

• Rapid protein production directly from DNA template

• Scalable protein production and automation (g to g amounts)

• Expression of difficult to express proteins (membrane proteins, toxic proteins)

• Studies on protein-protein, protein-DNA, or protein-RNA interactions

• Functional analysis of mutations, truncations, protein domains

• Protein engineering and use of artificial amino acids

• Protein labeling with stable isotopes (MS, NMR, crystallography)

• Production of antigens

~40% of all scientists in life science work on or otherwise use proteins for their studies!

Why Cell-Free Protein Synthesis?


Our Technology

CellFree Sciences holds IP on special production methods for high activity wheat germ extracts removing inhibiting activities from the seed:

Before washing steps After washing steps

0

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90

100

0 2 4 6 8 10 12 14

Yie

ld G

FP in

mg/

ml

Time in days(Data provided by Prof. Y. Endo)

“washed”

“unwashed”

8-10mg/mlGFP


Committed to Continuous Innovation

Publication Ehime University(new wheat germ extract preparation)2000

CFS releases WEPRO1240H/G(Optimized for His- and GTS-fusion proteins)2005

CFS releases WEPRO1240(Standard wheat germ extract)2004

CFS releases WEPRO7240 Series(High performance wheat germ extracts)2009

CFS releases WEPRO9240 Series(All-in-one ready setup reagents)2013


System Tag High Yield Low Yield Failed No Data

CFS Wheat germ (in vitro) 5’ GST 48 2 - -

CFS Wheat germ (in vitro) 3’ His 41 5 4 -

E. coli (in vitro) 5’ GST 28 2 20 -

E. coli (in vitro) 3’ His 6 - 42 2

E. coli (in vivo) 3’ His 18 13 18 1

DM2 Cells (cell culture) 3’ His 27 5 18 -

CHO Cells (cell culture) 3’ TAP 23 2 14 11

Protein expression test using 26 “soluble human proteins” and 24 “membrane human proteins”Table prepared using data from Supplementary Figure 3: “Comparison of activities of various protein expression systems”: N. Goshima et al., Nature Methods, 5, 1011-1017 (2008)

How does it compare?

Human Protein Factory compared expression of 50 proteins in different systems:


Products and Services for Protein Production

Providing complete solutions to serve customer needs

cDNA TemplatemRNA

Expression

Protein

Expression

Protein

Purification

Cloning services SP6 RNA polymerase Standard GST-tag

Gene synthesis RNase inhibitor High-Performance His-tag

PCR services NTP mix GST-tag Tag removal

Sequencing services His-tag

Vectors Protein labeling

Primers - Automated Protein Synthesizers -


pEU-E01 Vector Sets

SP6 promoter for in vitro transcription

E01 enhancer optimized for wheat germ system

Available for 5’ and 3’ tag fusion

Available for PreScisson and/or TEV protease

Do not require 5’ Cap structure or polyadenylation

(GST and His vectors are not offered in the USA and Canada until 2014) (DHFR = dihydrofolate reductase)

Standard Cloning His-TEVGST-TEV/GST-Pre Positive Control


High-Throughput Expression: Split-PCR

ORF5’ 3’Reverse Primer

Gene Primer

deSP6E01

SPU

ORFE01SP6

Allows for direct use of PCR products in protein synthesis!

ORF5’ 3’Reverse Primer

5’ 3’

1st PCR

2nd PCR

Split-Primer Pair

~ 1,500 bp


TranscriptionSP6 RNA polymerase

Translation

Linked Protein Expression Reactions

The wheat germ system uses separate transcription and translation reactions:

Optimal yields for each reaction means better overall protein yields!

Protein yields depend on RNA quality and amount.

http://www.google.co.jp/url?sa=i&rct=j&q=protein&source=images&cd=&cad=rja&docid=a4pmtA0pHGAddM&tbnid=rTgMkVDehQB1dM:&ved=0CAUQjRw&url=http://en.wikipedia.org/wiki/Protein&ei=Zwd6UZzqCcuXiQf0h4G4CA&bvm=bv.45645796,d.b2I&psig=AFQjCNEXlDFgq2YkR44pJE9-0xsjwuL1CQ&ust=1367038164117234


“Bilayer” Translation Reactions

Standard reaction format to allow for extended reaction times and higher yields:

Translation reaction can also be done by dialysis, or automated “Filter-Feed”.

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 4 8 12 16 20 24

16˚C Plasmid Bilayer 10 DO 16˚C Plasmid Batch 10 OD

26˚C Plasmid Bilayer 10 DO 26˚C Plasmid Batch 10 OD

Optimal reaction temperature at 16℃ for 24h reaction time and high yields.

RFU

Time


Dedicated Wheat Germ Extracts

Crude Protein Yields for Standard Expression Reactions

Application General Use H-Series G-Series Labeling

Product name: WEPRO1240 WEPRO1240H WEPRO1240G WEPRO2240(H)

Protein

analysis

“Standard” His-tag

Fusion Protein

For GST-tag

Fusion Protein

No amino acids

Product name: WEPRO7240 WEPRO7240H WEPRO7240G WEPRO8240(H)

Protein

production

“High

Performance”

His-tag

Fusion Protein

GST-tag

Fusion Protein

No amino acids

Reaction Size WEPRO1240 WEPRO7240 (WEPRO9240)

226 l 15 g 30 g 30 g

1,200 l 100 g 200 g 200 g

6,000 l 500 g 1,000 g 1,000 g

(Data for expression of GFP)


1: WEPRO® 1240 (Purified protein)

2: WEPRO® 7240 (Purified protein)

Up to two-fold higher yield for WEPRO7240!

Comparison WEPRO1240/WEPRO7240


Versions for His- and GST-tagged Proteins

Purified His-tagged protein Purified GST-tagged protein

S: WEPRO® 1240 H: WEPRO® 1240H G: WEPRO® 1240G

(kDa)

97

66

45

30

20

Purity 67% 82%

S G(kDa)

9766

45

30

20

Purity 30% 70%

S H

Optimal for the expression and purification of fusion proteins!

(Standard extract) (Pre-cleared extract for His) (Pre-cleared extract for GST)


Reagent Combination TableBilayer

Protemist DTII/DTII-S or ManualFilter-and-Feed

Protemist XE

Translation Machinery General Use Labeling General Use Labeling

WEPRO 1240 7240 2240 8240 7240 8240

Transcription Reagents

SP6 Polymerase & RNasin ✓ ✓ ✓ ✓ ✓ ✓

NTP Mix ✓ ✓ ✓ ✓ ✓ ✓

5x Transcription Buffer ✓ ✓

5x Transcription Buffer LM ✓ ✓ ✓ ✓

Translation Buffer

SUB-AMIX ✓

SUB-AMIX SGC ✓

SUB-AMIX NA (labeling) ✓

SUB-AMIX SGC NA (labeling) ✓

SUB-AMIX SG ✓

SUB-AMIX SG NA (labeling) ✓

Optimized Buffer Systems


Premium PLUS Entry Kits (WEPRO9240)

New WEPRO9240 Reagent Kits released in 2013!

CFS developed WEPRO9240 series to address common needs for protein research:

• Optimal for frequent protein synthesis (226 l reactions, up to 30 g protein)

• Cloning vector and Split PCR primers included

• Transcription reagents for 8 reactions included

• Translation reagents for 8 reactions included

• Control template pEU-E01-DHFR included

• Easy to use for setting up cell-free protein synthesis!


Premium PLUS Kit for MS

• Up to 99% labeling efficiency for incorporating [13C, 15N]-L-Lys and [13C, 15N]-L-Arg

• Wheat germ cell-free protein expression (WEPRO9240), proven for the synthesis of a wide range of proteins.

• Premixed reagents for rapid reaction setup of two-step process

• Kit includes cloning vector, primers, and positive control

Preparation of stable-isotope-labeled protein standards for quantitative mass spectrometry (SRM/MRM)

Transcription reaction

37℃, 1-4 hours

Translation reaction by Bilayer Method

16℃, 20-24 hours

DNA template

preparation in

pEU-E01 vector

DNA template

preparation by

split-primer

PCR method

or

Cambridge Isotope Laboratories, Inc.3 Highwood Drive, Tewksbury, MA, 01876, USA.

PH: 800.322.1174

Website: http://www.isotope.com


Scaling Cell-Free Protein Synthesis

WEPRO9240 Reaction Kits 226µl bilayer reactions: <30 µg

WEPRO7240 on Protemist® XE 10-100ml filter-feed reactions: <1g

WEPRO7240 on Protemist® DTII/DTII-S 6ml bilayer reactions: <1000 µg

New dialysis based ProductsAnd Application Protocols

Customer needs/applications

New dialysis based ProductsAnd Application Protocols

Customer needs/applications


Automated Protein Synthesizers

Bilayer

GenDecoder®

HTP Screening384 samples

Repeat Batch

Protemist®

Mass Production8 samples

Bilayer

Protemist® DT II/DT II-S

All-in-one Solution24 samples: Screening6 samples: Production

Filter-and-Feed(VivaFlow)

Protemist ® XE

Large-Scale Production1 sample

(sold out)

Operated for in-house protein synthesis services at CFS!


Protemist® DT II/DT II-S

Tabletop instrument for automated protein synthesis

Performs transcription and translation reactions

Can prepare 24 proteins in parallel on 1.2 ml scale (Protemist DTII only)

Can prepare 6 proteins in parallel on 6 ml scale

Purification of GST- and His-tagged proteins from 6 ml reactions

Used for protein screening and large-scale production

Running time: 35 h including protein purification

Used in CFS protein production services

1.2 ml Reactions: Up to 200 g crude protein

6 ml Reactions: Up to 1 mg crude protein

Up to 500 g purified protein (GST or His tag)


Protein Yield Purity

GFP 559 μg 85%

GUS 535 μg 82%

DHFR 549 μg 89%

P38 604 μg 87%

JNK2 490 μg 85%

M: molecular weight

marker

N: negative control

C: crude (3 μl)

E: eluate (0.5 μl)

(kDa)

20 -

25 -

30 -

40 -

50 -60 -

80 -100 -150 -250 -

C E

GFP BSA (ng)

50 100 250 500M N C E

GUS

C E

DHFR

C E

p38

C E

JNK2

(kDa)

20 -

25 -

30 -

40 -

50 -60 -

80 -100 -150 -250 -

C E

GFP BSA (ng)

50 100 250 500M N C E

GUS

C E

DHFR

C E

p38

C E

JNK2

Protein Yield Purity

GFP 510 μg 91%

GUS 377 μg 83%

DHFR 520 μg 91%

P38 551 μg 86%

JNK2 383 μg 82%

His-tag protein expression with WEPRO7240H

GST-tag protein expression with WEPRO7240G

M: molecular weight

marker

N: negative control

C: crude (3 μl)

E: eluate (0.5 μl)

Protemist® DT II/DT II-S: Example Data

Protemist DTII is used at CFS to produce >1,500 different proteins every year!


Protemist® XE

Large-scale automated protein synthesis

Using filter-feed method for reagent supply

Up to 10 mg crude protein per ml possible

Can perform 10 to 100 ml reactions

Run time from 24 to 48 h depending on scale

No automated protein purification

Used in CFS protein production services

10 ml Reaction: Up to 100 mg crude protein

100 ml Reaction: Up to 1 g crude protein


GenDecoder®

cDNA

Clone CollectionSplit-PCR

cDNA

TemplatesTranscription Translation

Protein

Samples

384 gene products/over night

Steps performed by GenDecoder®


High-Throughput Screening Assays

ScreeningSplit-PCR

GenDecoder®

(other robotic systems)

384 genes / day

cDNA Libraries

Clone Collections

Protein

Engineering

Protein Expression

384 proteins / day

(GenDecoder®)

384 reactions / few hours

(ALPHA screen™)

384 reactions / day

(FCS)

ALPHA screen™ and FCS analysis equipment are commercially available from PerkinElmer and Olympus respectively.

Feedback for evolutionary design

modification of desired proteins

Examples:

Protein kinase

・ALPHA screen™

Nuclear receptor

・FCS

Vaccine candidates

・ALPHA screen™

・FCS


Protein Production Services

Customer Question/Objective

Cloning resources for template generation

Cell free protein synthesis

QC and production report

Covering all steps from the DNA template to the expressed protein.

Cell-Free expression and automated protein synthesizers allow for fast turnaround times!


Customer Needs Template Preparation Protein Expression

Sequence data? Gene synthesis Solubility testing

cDNA clone? Sub-cloning cDNAs/ORFs Yield testing

cDNA fragments? Cloning of ORF regions QC report

Mutations? Introduction of mutations Optimization

Gene fusions? Cloning of fusion genes Up scaling production

Others… Split-PCR Purification

Inquire about Contract Research at CFS: Functional Protein Analysis and Screening Assay Development!

Protein Production Services

Template Preparation

Test Expression

Production on different scales

• 19,713 coding clones

• Each Clone unique

RefSeq ID

• 14,384 known genes

• 5,329 unknown genes

(as of Nov. 2012)


Annotated Clone Set: Human Gene and Protein Database (HGPD): http://hgpd.lifesciencedb.jp

Human ORF Clones

CFS licensed the right to use the AIST/JBIC Human ORF Clones for commercial purpose.

Input ID or keyword (e.g. “p53”)

→→ output: sequence, protein expression, solubility data, and so on.

“Nearly all proteins synthesized in the wheat germ cell-free expression system yield soluble fractions”

N. Goshima et al., Human protein factory for converting the transcriptome into an in vitro expressed proteome, Nature Methods, 5, 1011-1017 (2008)


Human Protein Active Array

• Specific interactions with ~14,000 human proteins can be screened by PAA

platform

• All human proteins are expressed at CFS our wheat germ system

• Use of GST-tag to immobilize proteins on magnetic beads

• Proteins stay in in solution at all times

• No protein denaturation during array production

• Detailed annotation data on human proteins available

PAA Screening Services are available for Contract Research at CFS.

Analyzing the human proteome using comprehensive AIST ORF cDNA collection:


Protein Active Array (PAA)

Assembled PAA PlateReaction plate

Magnetic plate

Protein Active Array (PAA) allows screening of entire Human ORF Clone set.

1,536 well format


PAA Standard Protocol

1. Incubate for 1 h with your antibody (concentration 1μg/ml) *2. Wash the plate with washing buffer3. Incubate for 1 h with 2nd antibody conjugated with horseradish peroxidase4. Wash the plate with washing buffer5. Incubate with chemiluminescence reagent6. Detect positive proteins by a high sensitive chemiluminescence analyzer

Reagent injection/incubation Detect positive wells by

Image analyzer

Image

Analyzer

Result (image)

* In total 400μg antibody required for entire profiling process


Chemiluminescence image data14 positive signals on array

(All human proteins are arrayed in duplicate)

Specific Profiling of anti-HA-tag mAB

HA-tag: Synthetic peptide from influenza hemagglutinin epitope: YPYDVPDYAScreening human protein set for reactivity to commercial anti-HA-tag monoclonal antibody

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

2201601201009080706050

40

30252015

Lane Entry_Clone_ID kDa

1 FLJ40958AAAF 52.06

2 FLJ10511AAAF 31.28

3 FLJ40876AAAF 25.49

4 FLJ40075AAAF 42.89

5 FLJ92933AAAF 29.41

6 FLJ20516AAAF 34.52

7 FLJ82655AAAF 89.09

8 FLJ10734AAAF 74.67

9 FLJ10882AAAF 68.62

10 FLJ81155AAAF 11.40

11 FLJ31145AAAF 41.99

12 FLJ54287AAAF 25.65

13 FLJ82936AAAF 133.56

14 FLJ83815AAAF 57.51

15 Negative Control

13 antigens out of 14 positive proteins were also detected by Western Blotting.


Detection of Autoantibodies in Cancer

Bladder

cancers

Lung

cancers

Detected only in lung cancers

Detected only in bladder cancersNormal

Detected in both cancers and normal

This research was supported by METI grant No.21R3007C.


Contract Research

CFS is an active partner working closely together with academic research

centers, as well as Biotech and Pharmaceutical Companies on protein related

joint research and development projects:

• Functional protein analysis

• High-throughput compound screening

• Assay development for individual proteins and applications

• New human Protein Active Arrays (PAA) for

Biomarker discovery

Profiling autoimmune antibodies

Antibody specificity screens

Contact us for more information on how we can support your R&D projects.


Basic to Technology (Description of the technology)Takai, T., Sawasaki, S., and Endo, Y. (2010) Practical cell-free protein synthesis system using purified wheat embryos, Nat. Protoc., 5, 227-238.

X-ray Crystallography (Using CellFree Sciences Products)Beebe, E.T., Makino, S., Nozawa, A., Matsubara, Y., Frederick, R.O., Primm, J.G., Goren, M.A., and Fox, B.G. (2010) Robotic large-scale application of wheat cell-free translation to structural studies including membrane proteins. New Biotechnol., 28, 239-249

Guild, K., Zhang, Y., Stacy, R., Mundt, E., Benbow, S., Green, A., and Myer, P.J. (2011) Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline. Acta Crystallogr. F., 67, 1027–1031

Mass Spectrometry (Using CellFree Sciences Products)Singh, S., Springer, M., Steen, J., Kirschner, M.W., and Steen, H. (2009) FLEXIQuant: A novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. J Proteome Res., 8, 2201 - 2210

Biotinylation (Using CellFree Sciences Products)Matsuoka, K., Komori, H., Nose, M., Endo, Y., and Sawasaki, T. (2010) Simple screening method for autoantigenproteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis. J Proteime Res., 9, 4264-4273

Selected References


iPS Cell Reseach (Using CellFree Sciences Products)Maekawa M, Yamaguchi K, Nakamura T, Shibukawa R, Kodanaka I, Ichisaka T, Kawamura Y, Mochizuki H, Goshima N, Yamanaka S. Direct reprogramming of somatic cells is promoted by maternal transcription factor Glis1. Nature. 2011 Jun 8;474(7350):225-9. doi: 10.1038/nature10106.

Expression of Membrane Proteins (Using CellFree Sciences Products)Nozawa, A., Ogasawara, T., Matsunaga, S., Iwasaki, T., Sawasaki, T., and Endo, Y. (2011) Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system. BMC Biotechnol., 11:35

Jarecki BW, Makino S, Beebe ET, Fox BG, Chanda B. Function of Shaker potassium channels produced by cell-free translation upon injection into Xenopus oocytes.Sci Rep. 2013;3:1040. doi: 10.1038/srep01040. Epub 2013 Jan 8.

Recent Reviews on Cell-Free Protein SynthesisCarlson ED, Gan R, Hodgman CE, Jewett MC. Cell-free protein synthesis: applications come of age.Biotechnol Adv. 2012 Sep-Oct;30(5):1185-94.

Casteleijn MG, Urtti A, Sarkhel S. Expression without boundaries: cell-free protein synthesis in pharmaceutical research. Int J Pharm. 2013 Jan 2;440(1):39-47. doi: 10.1016/j.ijpharm.2012.04.005. Epub 2012 Apr 11.

Selected References


Learn more about CellFree Sciences

Follow us on LinkedIn: http://www.linkedin.com/company/3139350?trk=tyah

Visit our homepage at: http://www.cfsciences.com/eg/index.html

Reagents for cell-free protein expression

Protein synthesizers

Protein expression services

Protein Active Array

Contract research for protein studies

7/18/2013 Matthias Harbers

Thank you for working with CFS!

38

Dr. Matthias Harbers

E-mail: [email protected]

Tel.:+81-45-500-2119 FAX:+81-45-500-2117

accelerating protein research

Technology

protein expression reactions

protein expression test

overall protein yields

rapid protein production

human protein factory

cellfree protein synthesis

artificial amino acids

data cfs wheat germ