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
10
20
30
40
50
60
70
80
90
100
0 2 4 6 8 10 12 14
YieldGFPinmg/ml
Time in days
(Data provided by Prof. Y. Endo)
“washed”
“unwashed”
8-10mg/ml
GFP

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 Template
mRNA
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

Transcription
SP6 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.

“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)
97
66
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 Table
Bilayer
Protemist DTII/DTII-S or Manual
Filter-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 Products
And Application Protocols
Customer needs/applications
New dialysis based Products
And Application Protocols
Customer needs/applications

Automated Protein Synthesizers
Bilayer
GenDecoder®
HTP Screening
384 samples
Repeat Batch
Protemist®
Mass Production
8 samples
Bilayer
Protemist® DT II/DT II-S
All-in-one Solution
24 samples: Screening
6 samples: Production
Filter-and-Feed
(VivaFlow)
Protemist ® XE
Large-Scale Production
1 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 Collection
Split-PCR
cDNA
Templates
Transcription 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 Plate
Reaction 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 buffer
3. Incubate for 1 h with 2nd antibody conjugated with horseradish peroxidase
4. Wash the plate with washing buffer
5. Incubate with chemiluminescence reagent
6. 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 data
14 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: YPYDVPDYA
Screening 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
220
160
120
100
90
80
70
60
50
40
30
25
20
15
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 autoantigen
proteins 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 Synthesis
Carlson 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: m-harbers@cfsciences.com
Tel.:+81-45-500-2119 FAX:+81-45-500-2117

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