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Products and Services Wheat Germ Cell-‐Free Protein Expression Introduction Cell-‐free protein expression or in vitro translation methods are used for a long time in protein analysis and studies on basic biochemical processes. With the great progress made over the last years, however, cell-‐free protein expression systems are available today that can cover all preparative needs for protein expression.Highly potent wheat germ extracts offer one of the most powerful and reliable cell-‐free protein expression system that is successfully used for the expression of individual proteins as well as large-‐scale genome-‐wide studies involving thousands of different proteins. The open nature of the wheat germ protein expression system is in particular useful for the preparation of isotope-‐labeled proteins and the preparation of proteoliposome complexes.On its own or in combination with isotope-‐labeled amino acids available from CIL/Euriso-‐top, wheat germ cell-‐free protein expression is the preferable approach to rapidly obtain the proteins at need to conduct your research. 1 | P a g e Protein Expression Made Easy CFS provides all reagents necessary to express your proteins of interest. Obtain Template > Express Protein > Confirm Expression! 2 | P a g e Applications • Rapid protein production directly from DNA template (vector or PCR product) • Scalable protein production and automation (µg to g amounts) • Expression of difficult to express proteins (membrane proteins, transcription factors, 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) • Rapid production of antigens for antibody production Linking RNA Expression to Protein Translation Two linked reactions offer optimal reaction conditions for both, RNA expression and protein translation. Note, many proteins are better expressed at temperatures below the optimal 37°C used in the RNA expression reactions. Different additives can be added to the protein translation reactions without interfering with RNA expression. 3 | P a g e Reaction Formats and Protein Yields Cell-‐free protein expression reactions can be performed in different reaction formats. While most protocols suggest easy-‐to-‐set-‐up batch reactions, those do not allow to maintain the translation reaction for more than a few hours. This may be sufficient for fast labeling reactions, but it comes at the expense of low protein yields and labeling efficiency. Therefore CFS uses different reaction formats to allow for extended reaction times and higher protein yields. Batch Bilayer Repeat Batch Filter-‐Feed Dialysis ~1.6 mg ~3.2 mg ~8 mg ~20 mg ~20 mg Relative amounts of Green Fluorescent Protein obtained from 1 ml WEPRO7240 wheat germ extract Bilayer reaction format Standard reaction format for small-‐scale or mid-‐range protein expression reactions. The wheat germ extract together with the RNA transcript is layered below the reaction buffer. The reaction buffer will feed thetranslation reaction for up to 24 h in a diffusion driven process. 4 | P a g e This reaction format is used in the CFS Premium PLUS Expression Kits and Protein Research Kits. Similarly, bilayer reactions can be fully automated as used on the Protemist DT II protein synthesizers. Dialysis Method For extended reaction times it is preferable to continuously supply the translation reactions with fresh substrates and to remove inhibitors of protein translation. This is best done by a dialysis method, where thetranslationreaction is performed in a dialysis cup or cassette. The dialysis cup or cassette is placed in a container with the reaction buffer. Using a membrane with a ~10,000 Da exclusion size, proteins and ribosomes will stay in the reaction mix while reaction byproducts and substrates can pass the dialysis membrane. Contact us for protocols for performing dialysis reactions on different reaction scales. 5 | P a g e Product Range CFS offers different products to meet with your protein production needs: • • • Premium PLUS Expression Kits and Protein Research Kits: 226 µl bilayer reaction scale Protein Expression Kits: 96 x 226 µl bilayer reaction scale 24 x 1200 µl bilayer reaction scale 6 x 6000 µl bilayer reaction scale Reagent Bulk Program: All reagents can be purchased individually to address the needs of large project. We can provide wheat germ extracts starting from 1 ml aliquots to liter amounts. 6 | P a g e Reagent Kits Premium PLUS Expression Kit (Starter Kit) Order Information: Premium PLUS Expression Kit CFS-‐EDX-‐PLUS 8 reactions -‐ 80 ℃ Kit contains reagents for 8 x 226 µl reactions. Shelf-‐life: 1 year Description: This starter kit is intended for testing the wheat germ cell-‐free expression system. The kit contains ready-‐to-‐use, premixed transcription and translation reagents including reaction vials for setting up 8 bilayer reactions on a 226 µl scale. The kit further provides the pEU expression vector optimized for the wheat germ expression system, a primer set for Split-‐PCR-‐based template preparation, a positive control, and a detailed manual. About 30 µg of crude protein can be obtained by use of the DHFR positive control gene provided with the kit. 7 | P a g e Kit Content: • pEU-‐E01-‐MCS vector • pEU-‐E01-‐DHFR vector (Positive control) • Primers for DNA preparation by PCR (SPU and deSP6E01) • Transcription premix LM • WEPRO®9240 • SUB-‐AMIX® SGC (Translation buffer) • Aluminum seals for reaction cups • Protocol booklet Protein Research Kits Order Information: Protein Research Kit (S)CFS-‐PRK-‐S2424 reactions -‐ 80 ℃ Protein Research Kit (G)CFS-‐PRK-‐G2424 reactions-‐ 80 ℃ Protein Research Kit (H)CFS-‐PRK-‐H2424 reactions-‐ 80 ℃ Each kit contains reagents for 24 x 226 µl reactions.Shelf-‐life: 1 year Description: 8 | P a g e Protein Research Kits provide ready-‐to-‐use premixed transcription and translation reagents and reaction cups for easy handling and setup of bilayer reactions on a 226 µl reaction scale. Special versions are available for the expression of GST-‐tag fusion proteins (G) and His-‐tag fusion proteins (H). For those Protein Research Kits the wheat germ extracts have been pre-‐cleared to reduce background binding during tag-‐mediated affinity purification. About 30 µg of crude protein can be obtained when using the DHFR positive control gene provided with the Premium PLUS Expression Kit. Kit Content: • Transcription premix LM • WEPRO®9240S/G/H • SUB-‐AMIX® SGC (Translation buffer) • Aluminum seals for reaction cups • Protocol booklet WEPRO 7240 Protein Expression Kits Order Information: WEPRO7240 Expression Kit CFS-‐TRI-‐7240 1 kit -‐ 80 ℃ WEPRO7240G Expression Kit CFS-‐TRI-‐7240G 1 kit -‐ 80 ℃ WEPRO7240H Expression Kit CFS-‐TRI-‐7240H 1 kit -‐ 80 ℃ Depending on reaction scale, each kit contains reagents for: 96 x 226 µl bilayer reaction scale 24 x 1200 µl bilayer reaction scale 6 x 6000 µl bilayer reaction scale Shelf-‐life: 1 year 9 | P a g e Description: WEPRO 7240 Protein Expression Kits provide the latest version of our most potent wheat germ cell-‐free expression system for general use in high yield protein synthesis. Each kit contains all the transcription and translation reagents for wheat germ cell-‐free protein synthesis where the reagents can be used to set up translation reactions on different reaction scales. Special versions are available for the expression of GST-‐tag fusion proteins (G) and His-‐tag fusion proteins (H). For those Protein Expression Kits the WEPRO 7240 wheat germ extracts have been pre-‐cleared to reduce background binding during tag-‐mediated affinity purification. About 30 µg of crude protein can be obtained when using the DHFR positive control gene in a 227 µl bilayer reaction. The Protein Expression Kits can be used on a Protemist DTII protein synthesizer. Kit Content: • • • • • • • • • • WEPRO7240S/G/H (240 OD, 2x1 ml) pEU-‐E01-‐MCS vector pEU-‐E01-‐DHFR vector (positive control vector) SP6 RNA Polymerase (80,000 units/ml, 30 µl) RNase Inhibitor (80,000 units/ml, 30 µl) NTP Mix (25 mM, 200 µl) 5x Transcription Buffer LM (400 µl) SUB-‐AMIX SGC (40x, 1.1 ml x 4) Creatine Kinase (20 mg/ml, single use only) Protocol booklet WEPRO 8240 Protein Expression Kits (Amino acid free) Order Information: 10 | P a g e WEPRO8240 Expression Kit CFS-‐TRI-‐8240 1 kit -‐ 80 ℃ WEPRO8240G Expression Kit CFS-‐TRI-‐8240G 1 kit -‐ 80 ℃ WEPRO8240H Expression Kit CFS-‐TRI-‐8240H 1 kit -‐ 80 ℃ Depending on reaction scale, each kit contains reagents for: 96 x 226 µl bilayer reaction scale 24 x 1200 µl bilayer reaction scale 6 x 6000 µl bilayer reaction scale Shelf-‐life: 1 year Description: WEPRO 8240 Protein Expression Kits provide amino acid free versions of our highly potent wheat germ cell-‐free expression system for effective protein labeling reactions. These kits should be used for heavy isotope labeling of proteins in combination with isotope-‐labeled amino acids sold separately by CIL/Euriso-‐top. Each kit contains all the transcription and translation reagents for wheat germ cell-‐free protein synthesis where the reagents can be used to set up translation reactions on different reaction scales. Special versions are available for the expression of GST-‐tag fusion proteins (G) and His-‐tag fusion proteins (H). For those Protein Expression Kits the WEPRO 8240 wheat germ extracts have been pre-‐cleared to reduce background binding during tag-‐mediated affinity purification. About 30 µg of crude protein can be obtained when using the DHFR positive control gene in a 227 µl bilayer reaction. Note, amino acids for conducting the translation reactions must be purchased separately. The Protein Expression Kits can be used on a Protemist DTII protein synthesizer. Kit Content: 11 | P a g e • • • • • • • • • • WEPRO8240S/G/H (240 OD, 2x1 ml) pEU-‐E01-‐MCS vector pEU-‐E01-‐DHFR vector (positive control vector) SP6 RNA Polymerase (80,000 units/ml, 30 µl) RNase Inhibitor (80,000 units/ml, 30 µl) NTP Mix (25 mM, 200 µl) 5x Transcription Buffer LM (400 µl) SUB-‐AMIX SGC NA (40x, 1.1 ml x 4) Creatine Kinase (20 mg/ml, single use only) Protocol booklet Protein Expression Kit for NMR Order Information: WEPRO8240 (15N, 13C) Expression Kit TRI-‐8240S15N13C 1 kit -‐ 80 ℃ Depending on reaction scale, each kit contains reagents for: 96 x 226 µl bilayer reaction scale 24 x 1200 µl bilayer reaction scale 6 x 6000 µl bilayer reaction scale Shelf-‐life: 1 year Description: The WEPRO 8240 Protein Expression Kit for NMR provides15N, 13C-‐labeled amino acidsforthe preparation of isotope-‐labeled proteins for NMR studies. The kit contains all the transcription and translation reagents for wheat germ cell-‐free protein synthesis where the reagents can be used to set up translation reactions on different reaction scales. Special version are available on customer request for the expression of GST-‐tag fusion proteins (G) and His-‐tag fusion proteins (H). For those Protein Expression Kits the WEPRO 8240 wheat germ extracts have been pre-‐ cleared to reduce background binding during tag-‐mediated affinity purification. About 30 µg of 12 | P a g e crude protein can be obtained when using the DHFR positive control gene in a 227 µl bilayer reaction. The Protein Expression Kits can be used on a Protemist DTII protein synthesizer. Kit Content: ・ ・ ・ ・ ・ ・ ・ ・ ・ ・ WEPRO8240 (240 OD, 1 ml x 2), store at -‐80˚C SUB-‐AMIX SGC (15N, 13C)(S1~S4, 1.1 ml x 4) SP6 RNA Polymerase (80,000 unit/ml, 30 μl) RNase Inhibitor (80,000 unit/ml, 30 μl) 5x Transcription Buffer LM (400 μl) NTP Mix (25 mM, 200 μl) Creatine Kinase (20 μl) (single use only) pEU-‐E01-‐MCS vector pEU-‐E01-‐DHFR vector (positive control) Protocol booklet Premium PLUS Expression Kit for MS Order Information: Premium PLUS Expression Kit for MS CFS-‐EDX-‐PLUS-‐MS 16 reactions -‐ 80 ℃ Kit contains reagents for 16 x 226 µl reactions.Shelf-‐life: 1 year Description: This Premium PLUS Expression Kit for MS was optimized for very high incorporation rates of [13C, 15 N]-‐L-‐Lys and [13C, 15N]-‐L-‐Arg for use in protein MS studies. The kit contains ready-‐to-‐use, premixed transcription and translation reagents including [13C, 15N]-‐L-‐Lys and [13C, 15N]-‐L-‐Arg to set up 16 bilayer reactions on a 226 µl scale. The kit further provides the pEU expression vector 13 | P a g e optimized for the wheat germ expression system, a primer set for Split-‐PCR-‐based template preparation, a positive control, and a detailed manual. About 30 µg of crude protein can be obtained by use of the DHFR positive control gene provided with the kitachieving 99% labeling efficiency. Inquire for special vectors for His-‐tagged fusion protein not included in the kit. Using a modified primer not included in the kit, templates for the preparation of His-‐tagged proteins can also be directly prepared by PCR. Kit Content: • • • • • • • • pEU-‐E01-‐MCS vector (expression vector) pEU-‐E01-‐DHFR vector (positive control) PCR primer set (SPU, deSP6E01) Transcription Premix LM WEPRO9240 for MS SUB-‐AMIX SGC for MS (provided in reaction cups ready to use) Aluminum seals for reaction cups Protocol booklet 14 | P a g e Cell-‐Free Protein Expression – Teaching Kit Order Information: Protein Expression Reagent KitCFS-‐EDU 10 assays -‐ 80 ℃ Laboratory Instrument Set CFS-‐EDU-‐E for 10 groups RT Shelf-‐life: 1 year Description: The Cell-‐Free Protein Expression Teaching Kit provides reagents for 10 protein expression experiments intended for the education of 20 students per class. During the experiment, a Green Fluorescent Protein will be synthesized that can be easily detected by the students. The controls included into the experiment explain the need to first prepare an mRNA transcript from a DNA template that is then translated into the protein. UV lamps for detecting the protein can be purchased separately. Protein Expression Reagent Kit Content: • • • • • • • • • • Distilled water Transcription buffer Ribonucleotides 10mg/mL RNase 10,000 unit/mL RNase inhibitor 10,000 unit/mL RNA polymerase 1 mg/mL Plasmid DNA Fluorescent detection reagent (Not including in Kit* for sales outside of Japan) Wheat germ extract Protocol booklet Laboratory Instrument Set content: • • Tube rack Floater 15 | P a g e • • • Orangefilter BlueLED light UV – LED light Reagents for RNA Expression and Protein Translation Reagents for in vitro transcription reactions Product Name Product Number Description SP6 RNA polymerase Enzyme for mRNA synthesis and CFS-‐TSC-‐ENZ plus RNase inhibitor inhibitor of RNase activity SP6 RNA polymerase plus RNase inhibitor CFS-‐TSC-‐ENZ-‐S Enzyme for mRNA synthesis and inhibitor of RNase activity NTP Mix CFS-‐TSC-‐NTP Mixture of ATP, UTP, GTP, and CTP 5xTranscripton Buffer Transcription buffer for CFS-‐TSC-‐5TB-‐LM LM WEPRO7240/8240 series High-‐Performance Wheat Germ in vitro translation reagents Product Name Product Number Description WEPRO7240 CFS-‐WGE-‐7240 High performance wheat germ extract Pre-‐processed, high performance WEPRO7240G CFS-‐WGE-‐7240G wheat germ extract for high purity collection of GST-‐tagged proteins WEPRO7240H WEPRO8240 WEPRO8240G WEPRO8240H SUB-‐AMIX SGC SUB-‐AMIX SGC NA 16 | P a g e Pre-‐processed, high performance wheat germ extract for high purity collection of His-‐tagged proteins High performance wheat germ extract for labeling, containing only traces of CFS-‐WGE-‐8240 amino acids; WEPRO7240 less amino acids High performance wheat germ extract for labeling, containing only traces of CFS-‐WGE-‐8240G amino acids, pre-‐processed for His tag affinity purification; WEPRO8240G less amino acids High performance wheat germ extract for labeling, containing only traces of CFS-‐WGE-‐8240H amino acids, pre-‐processed for His tag affinity purification; WEPRO8240H less amino acids For Bilayer translation reactions Translation buffer; to be used with CFS-‐SUB-‐SGC WEPRO7240 series Translation buffer for labeling; SUB-‐ CFS-‐SUB-‐SGC-‐ AMIX SGC less amino acids; to be used NAA with WEPRO8240 series For Filter-‐Feed translation reactions for Protemist XE CFS-‐WGE-‐7240H Specification 80,000 units 1 ml 2,400 units 30 µl 1 ml 1 ml Specification 1 ml 1 ml 1 ml 1 ml 1 ml 1 ml 12.5 ml x 4 12.5 ml x 4 SUB-‐AMIX SG SUB-‐AMIX SG NA Translation buffer; to be used with WEPRO7240 series Translation buffer for labeling; SUB-‐ CFS-‐SUB-‐SG-‐NAA AMIX SG less amino acids; to be used with WEPRO8240 series CFS-‐SUB-‐SG 12.5 ml x 4 12.5 ml x 4 Consumables for Protein Synthesizers Protemist® DT II Consumables Product Name Product Number Reaction cups CFS-‐DT2-‐P01-‐1 Reaction cups CFS-‐DT2-‐P01-‐10 Reaction cups CFS-‐DT2-‐P01-‐50 CFS-‐ASP-‐ Robotic tips, Bulk DIP1000V CFS-‐ASP-‐ Robotic tips, Racked DIP1000V-‐PT24 Tubes CFS-‐DT2-‐P02-‐2 Protemist® XE Consumables Product Name Product Number Pre-‐assembled Kit CFS-‐XE-‐P10 Pre-‐assembled Kit CFS-‐XE-‐P20 Connectors CFS-‐XE-‐VFC Description 6 x Disposable reaction cups 60 x Disposable reaction cups 300 x Disposable reaction cups 1000 x Disposable pipetting tips 24 x Ready to use disposable pipetting tips in 96-‐well racks 1000 x 2 ml tubes Description 10 x 10 ml reactions 10 x 20, 30, 40 ml reactions 24 x Vivaflow Connector (needed for 30 and 40 ml reactions) Services Cell-‐Free Sciences provides template preparation and protein expression services based on the wheat germ cell-‐free protein expression system. 17 | P a g e Inquire on using our services for testing our cell-‐free protein expression system and customized protein preparations. We can also prepare on request expression templates for the wheat germ system. Automated Protein Synthesizers Protemist® DT II Robotic Protein Synthesizer Instrument Description: ・ Wheat germ cell-‐free protein synthesis ・ Fully automated transcription, translation, and purification in less than 35 hours ・ Two reaction sizes possible by simple cartridge exchange ・ Per run up to 6 x 6ml or 24 x 1.2ml translation reactions ・ Batch protein affinity purification in 6-‐ml translation format ・ Disposable reaction containers ・ Controlled temperature range for protein synthesis and purification ・ Instrument provided with full control and monitoring software and computer 18 | P a g e Instrument Specifications: Synthesis method ENDEXT® cell-‐free protein synthesis technology; Bilayer continuous diffusion method Max. number of samples 6 samples: transcription, translation (6 ml/sample), per run affinity purification 24 samples: transcription, translation (1.2 ml/sample) Reagent unit ・Temperature control method Peltier module 4℃ (Fixed) ・Temperature setting Reaction unit ・Temperature control method ・Temperature control Peltier module 4～37℃ Within ±1.5℃ range ・Temperature control variation PC (external) ・CPU Operational control & display IntelR CeleronR M Processor (1.50 GHz) 37 GB 19 | P a g e ・HDD ・Memory 256 MB Windows XP (English version) 14.1 inch ・OS ・Monitor Dispensing volume variation Ambience for installation Power supply ・Voltage fluctuation tolerance Within ±5%, CV < 1% (in dispensing 100 µl distilled water) 15～30℃, humidity < 75%, dew condensation to be avoided AC 100-‐240 V ±10% 350 VA ・Power consumption Dimensions and weight (main unit) Instrument Consumables: ・ Robotic tips ・ Reaction cups ・ 2 ml tube ・ 14 ml tube ・ 50 ml tube ・ PCR tube 20 | P a g e W: 700 mm (28"), H: 790 mm (31"), D: 500 mm (20"); 100 kg (220 lb) ・ 24 well plate Instrument Reagents: CFS provides different Expression Kits containing all reagents for one run with 6 x 6ml or 24 x 1.2ml reactions. Note, our Expression Kits do not include reagents for protein purification. Sample Requirements: Purified template DNA in vectors of the pEU-‐E01 family at a concentration of 1µg/µl. Standard Protein Yields: Determined by expression of GFP: ・ 1 mg crude protein in 6 ml reaction ・ 0.2 mg crude protein in 1.2 ml reaction ・ 0.5 mg purified protein for use of His or GST tag and 6 ml reaction Warranty: CFS gives a one-‐year warranty on each instrument from the date of instrument acceptance by customer. Protemist® XE Protein Synthesizer Instrument Description: ・ Wheat germ cell-‐free protein synthesis ・ Fully automated 24-‐ to 48-‐hour protein synthesis for 1 sample ・ Continual filter-‐and-‐feed translation through intermittent replenishment of substrate buffer and mRNA ・ About 10 mg crude protein per ml reaction volume 21 | P a g e ・ Variable reaction scale ranging from 10 ml to 100 ml Instrument Specifications: Synthesis method Number of sample per run Translation reaction volume Pump Filter unit PC (external) ENDEXT® cell-‐free protein synthesis technology; Filter-‐and-‐Feed translation reaction 1 sample 10, 20, 30, 40 ml Peristaltic pump (Masterflex®) Vivaflow 50 (Sartorius) Operational control & display ・CPU Intel® Celeron® 585 Processor (2.16 GHz) ・RAM 1 GB ・OS ・Monitor Ambience (inside an incubator) 22 | P a g e Windows Vista Business 14.1 inch 15～40℃, humidity < 85%, dew condensation to be avoided Power supply AC 100-‐240 V Dimensions and weight W: 525 mm (21"), H: 495 mm (19"), D: 310 mm (12"); (main unit) 20 kg (44 lb) Instrument Consumables: ・ Vivaflow 50 ・ Tubing harness set ・ 50 ml tube ・ Stericup bottle Instrument Reagents: No dedicated reagent kits, purchase reagents in bulk. Sample Requirements: Prepare mRNA from template DNA in vectors of the pEU-‐E01 family. Standard Protein Yields: Determined by expression of GFP: ・ 10 mg/ml translation reaction crude protein ・ Range ~100 mg for 10 ml reaction to ~1,000 mg for 100 ml reaction Warranty: CFS gives a one-‐year warranty on each instrument from the date of instrument acceptance by customer. 23 | P a g e Vectors Contact Cell-‐Free Sciences for obtaining expression vectors for the wheat germ cell-‐free expression system. Those vectors contain an optimized enhancer for high protein yields specifically selected for use in our expression system. Standard vector including positive controls pEU-‐E01-‐MCS pEU-‐E01-‐DHFR His-‐tag vector set for use of TEV protease (not Canada) pEU-‐E01-‐His-‐TEV-‐MCS-‐N1 pEU-‐E01-‐His-‐TEV-‐MCS-‐N2 pEU-‐E01-‐His-‐TEV-‐MCS-‐N3 pEU-‐E01-‐His-‐TEV-‐MCS-‐C1 pEU-‐E01-‐His-‐TEV-‐MCS-‐C2 pEU-‐E01-‐His-‐TEV-‐MCS-‐C3 GST-‐tag vector set for use of TEV protease (not USA and Canada) pEU-‐E01-‐GST-‐TEV-‐MCS-‐N1 pEU-‐E01-‐ GST -‐TEV-‐MCS-‐N2 pEU-‐E01-‐ GST -‐TEV-‐MCS-‐N3 pEU-‐E01-‐ GST -‐TEV-‐MCS-‐C1 pEU-‐E01-‐ GST -‐TEV-‐MCS-‐C2 Selected References On the wheat germ cell-‐free protein expression system: Wheat germ systems for cell-‐free protein expression. Harbers M., FEBS Lett. 2014 Jun 12. pii: S0014-‐5793(14)00471-‐2. doi: 10.1016/j.febslet.2014.05.061. Practical cell-‐free protein synthesis system using purified wheat embryos. Takai K, Sawasaki T, Endo Y., Nat Protoc. 2010 Feb;5(2):227-‐38. Structural genomics: 24 | P a g e Automated cell-‐free protein production methods for structural studies, Methods Beebe ET, Makino S, Markley JL, Fox BG. 2014 Mol Biol. 2014;1140:117-‐35. doi: 10.1007/978-‐1-‐ 4939-‐0354-‐2_9. Cell-‐free protein synthesis for functional and structural studies. Makino, S., Beebe, E., Markley, J., and Fox, B. Methods in Molecular Biology2014, 1091, 161-‐78. Wheat germ cell-‐free platform for eukaryotic protein production Vinarov, D., Loushin Newman,C., and Markley, J. 2006, FEBS J, 273, 4160-‐4169. Cell-‐free protein production and labeling protocol for NMR-‐based structural proteomics. Vinarov, D., Lytle, B., Peterson, F., Tyler, E., Volkman, B., and Markley, J. 2004Nat Methods 1 (2), 149-‐153. Protein engineering and screening: Cell-‐Free Protein Synthesis: Methods and Protocols Takasuka TE, Walker JA, Bergeman LF, Vander Meulen KA, Makino S, Elsen NL, and Fox BG. 2014 “Cell-‐free translation of biofuel enzymes”, Kirill Alexandrov and Wayne A. Johnston (ed.), Methods in Molecular Biology, Vol. 1118, 71-‐95. Protein MS: FLEXIQuant: A novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. Singh, S., Springer, M., Steen, J., Kirschner, M.W., and Steen, H. (2009) J Proteome Res., 8, 2201 – 2210 FLEXIQinase, a mass spectrometry-‐based assay, to unveil multikinase mechanisms. Singh SA, Winter D, Bilimoria PM, Bonni A, Steen H, and Steen JA. (2012) Nat Methods. Apr 8;9(5):504-‐8. doi: 10.1038/nmeth.1970. A practical guide to the FLEXIQuant method. Singh S, Kirchner M, Steen JA, Steen H., Methods Mol Biol. 2012;893:295-‐319. doi: 10.1007/978-‐1-‐ 61779-‐885-‐6_19. Expression of membrane proteins: Modifications of wheat germ cell-‐free system for functional proteomics of plant membrane proteins. Nozawa A, and Tozawa Y. 2014Methods Mol Biol. 1072, 259-‐72. Cell-‐free protein synthesis of membrane (1,3)-‐β-‐d-‐glucan (curdlan) synthase: co-‐translational insertion in liposomes and reconstitution in nanodiscs. Periasamy A, Shadiac N, Amalraj A, Garajová S, Nagarajan Y, Waters S, Mertens HD, and Hrmova M. 2013Biochim Biophys Acta. 1828(2), 743-‐57. 25 | P a g e Function of Shaker potassium channels produced by cell-‐free translation upon injection into Xenopus oocytes. Jarecki BW, Makino S, Beebe ET, Fox BG, and Chanda B. 2013Sci Rep. 3,1040. A cell-‐free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters. Norawa A, Nanamlya H, Mlyata T, Linka N, Endo Y,Weber AP, and Tozawa Y. 2007Plant Cell Physiol, 48,1815-‐1820. Genome-‐wide studies: Profiling of autoantibodies in sera of pancreatic cancer patients. Nagayoshi Y, Nakamura M, Matsuoka K, Ohtsuka T, Mori Y, Kono H, Aso T, Ideno N, Takahata S, Ryo A, Takeda H, Ito T, Oda Y, Endo Y, Sawasaki T, Tanaka M. Ann Surg Oncol. 2014 Jun;21 Suppl 3:S459-‐65. doi: 10.1245/s10434-‐014-‐3574-‐0. Epub 2014 Mar 1. Human protein factory for converting the transcriptome into an in vitro-‐expressed proteome. Goshima N et al. Nat Methods. 2008 Dec;5(12):1011-‐7. ESPRESSO: a system for estimating protein expression and solubility in protein expression systems. Hirose S, Noguchi T. Proteomics. 2013 May;13(9):1444-‐56. Vaccine Development: Wheatgerm cell-‐free system-‐based production of hemagglutinin-‐neuraminidase glycoprotein of human parainfluenza virus type 3 for generation and characterization of monoclonal antibody. Matsunaga S, Kawakami S, Matsuo I, Okayama A, Tsukagoshi H, Kudoh A, Matsushima Y, Shimizu H, Okabe N, Hirano H, Yamamoto N, Kimura H, Ryo A. Front Microbiol. 2014 May 13;5:208. doi: 10.3389/fmicb.2014.00208. eCollection 2014. Application of wheatgermcell-‐free protein expression system for novel malaria vaccine candidate discovery. Arumugam TU, Ito D, Takashima E, Tachibana M, Ishino T, Torii M, Tsuboi T. Expert Rev Vaccines. 2014 Jan;13(1):75-‐85. doi: 10.1586/14760584.2014.861747. Epub 2013 Dec 5. 26 | P a g e Downloads Flyers Manuals Vector Maps Showcases 8/14 Supersedes all previously published literature BNMR_CFS_WGCFPE 27 | P a g e
