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7/18/2013 Matthias Harbers 1
Dr. Matthias Harbers – Vice President Sales and Marketing Division
Accelerating Protein Resea...
7/18/2013 Matthias Harbers 2
CellFree Sciences offers a cell-free protein expression platform
technology to rapidly delive...
7/18/2013 Matthias Harbers 3
The Company
CellFree Sciences Co., Ltd.
 Based on ENDEXT® Technology
developed in the labora...
7/18/2013 Matthias Harbers 4
Cell-free protein synthesis is the preferable protein preparation method for:
• Rapid protein...
7/18/2013 Matthias Harbers 5
Our Technology
CellFree Sciences holds IP on special production methods for high activity
whe...
7/18/2013 Matthias Harbers 6
Committed to Continuous Innovation
Publication Ehime University
(new wheat germ extract prepa...
7/18/2013 Matthias Harbers 7
System Tag High Yield Low Yield Failed No Data
CFS Wheat germ (in vitro) 5’ GST 48 2 - -
CFS ...
7/18/2013 Matthias Harbers 8
Products and Services for Protein Production
Providing complete solutions to serve customer n...
7/18/2013 Matthias Harbers 9
pEU-E01 Vector Sets
 SP6 promoter for in vitro transcription
 E01 enhancer optimized for wh...
7/18/2013 Matthias Harbers 10
High-Throughput Expression: Split-PCR
ORF5’ 3’
Reverse Primer
Gene Primer
deSP6E01
SPU
ORFE0...
7/18/2013 Matthias Harbers 11
Transcription
SP6 RNA polymerase
Translation
Linked Protein Expression Reactions
The wheat g...
7/18/2013 Matthias Harbers 12
“Bilayer” Translation Reactions
Standard reaction format to allow for extended reaction time...
7/18/2013 Matthias Harbers 13
Dedicated Wheat Germ Extracts
Crude Protein Yields for Standard Expression Reactions
Applica...
7/18/2013 Matthias Harbers 14
1: WEPRO® 1240 (Purified protein)
2: WEPRO® 7240 (Purified protein)
Up to two-fold higher yi...
7/18/2013 Matthias Harbers 15
Versions for His- and GST-tagged Proteins
Purified His-tagged protein Purified GST-tagged pr...
7/18/2013 Matthias Harbers 16
Reagent Combination Table
Bilayer
Protemist DTII/DTII-S or Manual
Filter-and-Feed
Protemist ...
7/18/2013 Matthias Harbers 17
Premium PLUS Entry Kits (WEPRO9240)
New WEPRO9240 Reagent Kits released in 2013!
CFS develop...
7/18/2013 Matthias Harbers 18
Premium PLUS Kit for MS
• Up to 99% labeling efficiency for
incorporating [13C, 15N]-L-Lys a...
7/18/2013 Matthias Harbers 19
Scaling Cell-Free Protein Synthesis
WEPRO9240 Reaction Kits 226µl bilayer reactions: <30 µg
...
7/18/2013 Matthias Harbers 20
Automated Protein Synthesizers
Bilayer
GenDecoder®
HTP Screening
384 samples
Repeat Batch
Pr...
7/18/2013 Matthias Harbers 21
Protemist® DT II/DT II-S
 Tabletop instrument for automated protein synthesis
 Performs tr...
7/18/2013 Matthias Harbers 22
Protein Yield Purity
GFP 559 μg 85%
GUS 535 μg 82%
DHFR 549 μg 89%
P38 604 μg 87%
JNK2 490 μ...
7/18/2013 Matthias Harbers 23
Protemist® XE
 Large-scale automated protein synthesis
 Using filter-feed method for reage...
7/18/2013 Matthias Harbers 24
GenDecoder®
cDNA
Clone Collection
Split-PCR
cDNA
Templates
Transcription Translation
Protein...
7/18/2013 Matthias Harbers 25
High-Throughput Screening Assays
ScreeningSplit-PCR
GenDecoder®
(other robotic systems)
384 ...
7/18/2013 Matthias Harbers 26
Protein Production Services
Customer Question/Objective
Cloning resources for template gener...
7/18/2013 Matthias Harbers 27
Customer Needs Template Preparation Protein Expression
Sequence data? Gene synthesis Solubil...
• 19,713 coding clones
• Each Clone unique
RefSeq ID
• 14,384 known genes
• 5,329 unknown genes
(as of Nov. 2012)
7/18/201...
7/18/2013 Matthias Harbers 29
Human Protein Active Array
• Specific interactions with ~14,000 human proteins can be screen...
7/18/2013 Matthias Harbers 30
Protein Active Array (PAA)
Assembled PAA Plate
Reaction plate
Magnetic plate
Protein Active ...
7/18/2013 Matthias Harbers 31
PAA Standard Protocol
1. Incubate for 1 h with your antibody (concentration 1μg/ml) *
2. Was...
7/18/2013 Matthias Harbers 32
Chemiluminescence image data
14 positive signals on array
(All human proteins are arrayed in...
7/18/2013 Matthias Harbers 33
Detection of Autoantibodies in Cancer
Bladder
cancers
Lung
cancers
Detected only in lung can...
7/18/2013 Matthias Harbers 34
Contract Research
CFS is an active partner working closely together with academic research
c...
7/18/2013 Matthias Harbers 35
Basic to Technology (Description of the technology)
Takai, T., Sawasaki, S., and Endo, Y. (2...
7/18/2013 Matthias Harbers 36
iPS Cell Reseach (Using CellFree Sciences Products)
Maekawa M, Yamaguchi K, Nakamura T, Shib...
7/18/2013 Matthias Harbers 37
Learn more about CellFree Sciences
Follow us on LinkedIn: http://www.linkedin.com/company/31...
7/18/2013 Matthias Harbers
Thank you for working with CFS!
38
Dr. Matthias Harbers
E-mail: m-harbers@cfsciences.com
Tel.:+...
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Accelerating Protein Research

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Learn more about CellFree Sciences and our products and services for cell-free protein synthesis.

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Transcript of "Accelerating Protein Research"

  1. 1. 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
  2. 2. 7/18/2013 Matthias Harbers 2 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!
  3. 3. 7/18/2013 Matthias Harbers 3 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
  4. 4. 7/18/2013 Matthias Harbers 4 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?
  5. 5. 7/18/2013 Matthias Harbers 5 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
  6. 6. 7/18/2013 Matthias Harbers 6 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
  7. 7. 7/18/2013 Matthias Harbers 7 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:
  8. 8. 7/18/2013 Matthias Harbers 8 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 -
  9. 9. 7/18/2013 Matthias Harbers 9 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
  10. 10. 7/18/2013 Matthias Harbers 10 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
  11. 11. 7/18/2013 Matthias Harbers 11 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.
  12. 12. 7/18/2013 Matthias Harbers 12 “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
  13. 13. 7/18/2013 Matthias Harbers 13 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)
  14. 14. 7/18/2013 Matthias Harbers 14 1: WEPRO® 1240 (Purified protein) 2: WEPRO® 7240 (Purified protein) Up to two-fold higher yield for WEPRO7240! Comparison WEPRO1240/WEPRO7240
  15. 15. 7/18/2013 Matthias Harbers 15 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)
  16. 16. 7/18/2013 Matthias Harbers 16 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
  17. 17. 7/18/2013 Matthias Harbers 17 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!
  18. 18. 7/18/2013 Matthias Harbers 18 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
  19. 19. 7/18/2013 Matthias Harbers 19 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
  20. 20. 7/18/2013 Matthias Harbers 20 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!
  21. 21. 7/18/2013 Matthias Harbers 21 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)
  22. 22. 7/18/2013 Matthias Harbers 22 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!
  23. 23. 7/18/2013 Matthias Harbers 23 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
  24. 24. 7/18/2013 Matthias Harbers 24 GenDecoder® cDNA Clone Collection Split-PCR cDNA Templates Transcription Translation Protein Samples 384 gene products/over night Steps performed by GenDecoder®
  25. 25. 7/18/2013 Matthias Harbers 25 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
  26. 26. 7/18/2013 Matthias Harbers 26 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!
  27. 27. 7/18/2013 Matthias Harbers 27 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
  28. 28. • 19,713 coding clones • Each Clone unique RefSeq ID • 14,384 known genes • 5,329 unknown genes (as of Nov. 2012) 7/18/2013 Matthias Harbers 28 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)
  29. 29. 7/18/2013 Matthias Harbers 29 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:
  30. 30. 7/18/2013 Matthias Harbers 30 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
  31. 31. 7/18/2013 Matthias Harbers 31 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
  32. 32. 7/18/2013 Matthias Harbers 32 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.
  33. 33. 7/18/2013 Matthias Harbers 33 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.
  34. 34. 7/18/2013 Matthias Harbers 34 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.
  35. 35. 7/18/2013 Matthias Harbers 35 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
  36. 36. 7/18/2013 Matthias Harbers 36 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
  37. 37. 7/18/2013 Matthias Harbers 37 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
  38. 38. 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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