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Reference List

Selected References for Wheat Germ Cell-Free Protein Expression


The wheat germ cell-free protein expression system has been used for many years as a basic tool to drive life science research and to support product development. Below some selected references are provided to give examples on how the system had been used to address specific protein needs.

Search condition


The wheat germ cell-free protein expression system developed at Ehime University has been described in detail in the following publications:


A cell-free protein synthesis system for high-throughput proteomics.

Sawasaki T. et al.: Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14652-7.

PMID: 12409616 PMCID: PMC137474

・Original publication of the wheat germ cell-free expression system developed by the Endo laboratory

・Split-PCR protocol to prepare linear expression templates by two-step PCR method for direct use in cell-free protein expression


Practical cell-free protein synthesis system using purified wheat embryos.

Takai K., Sawasaki T., Endo Y.: Nat Protoc. 2010 Feb;5(2):227-38.

PMID: 20134421

Detailed protocol on how to prepare highly active wheat germ extracts for translation experiments



More information on the widespread use of the wheat germ cell-free protein expression systems can be found in the following review articles:


The wheat-germ cell-free expression system.

Takai K, Sawasaki T, Endo Y.: Curr Pharm Biotechnol. 2010 Apr;11(3):272-8.

PMID: 20210744


Wheat germ systems for cell-free protein expression.

Harbers M.: FEBS Lett. 2014 Aug 25;588(17):2762-73.

PMID: 24931374

Template Preparation

In cell-free protein expression systems, expression vectors and linear DNA can be used as templates to guide protein expression. For the wheat germ system, a special enhancer has been developed that induces translation without need for a Kozak consensus sequence. The E01 enhancer is used in all expression vectors from CFS:


Selection of 5'-untranslated sequences that enhance initiation of translation in a cell-free protein synthesis system from wheat embryos.

Kamura N. et al.: Bioorg Med Chem Lett. 2005 Dec 15;15(24):5402-6.

PMID: 16213724

・Screening random libraries of mRNA 5'-leader sequences to find new translation initiation site for wheat germ system

・Comparing activity to Omega sequence from tobacco mosaic virus


Additional expression vectors using the E01 enhancer have been described in the literature for working with different affinity tags:


A novel family of expression vectors with multiple affinity tags for wheat germ cell-free protein expression.

Nagy S.K. et al.: BMC Biotechnol. 2020 Mar 14;20(1):17.

PMID: 32169064, PMCID: PMC7071761

・pEU3-NII vector with rare-cutting NotI restriction enzyme cleavage site for vector linearization

・Vectors for using His12, FLAG, and Halo-tag

・GST-His6 and GST-biotin double-tagging vectors

Bilayer Reaction Format

To increase the protein yields for cell-free protein expression reactions, Ehime University developed the bilayer reaction format for the wheat germ cell-free expression system. This reaction format is used in several of our reagent kits:


A bilayer cell-free protein synthesis system for high-throughput screening of gene products.

Sawasaki T. et al.: FEBS Lett. 2002 Mar 6;514(1):102-5.

PMID: 11904190

・Diffusion system in bilayer setup enables the continuous supply of substrates

・Prolonged reaction times yield in more than 10 times more protein than in similar batch-mode reactions

Protein MS and Proteomics

Protein mass spectrometry has become a very powerful method to analyze individual proteins or even complex protein mixtures from clinical or biological samples. Protein quantification in such samples is often done by adding a reference peptide or reference protein having a higher mass than the native protein. This mass-shift is commonly achieved by incorporation of isotope-labeled amino acid(s) or a chemical modification.  The wheat germ cell-free protein expression system has been used to prepare such protein standards by direct incorporation of labeled amino acids, or chemical modification after completion of protein synthesis. CFS supports the use of our system in protein MS by providing dedicated reagents to prepare 13C/15N lysine and arginine reference proteins:


High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.

Takemori N. et al.: Mol Biosyst. 2015 Feb;11(2):361-5.

PMID: 25431973

・High throughout expression of membrane proteins into proteoliposomes

・High incorporation rates for added isotope-labeled amino acids

・Use as internal standard library for targeted transmembrane proteomics


A large-scale targeted proteomics assay resource based on an in vitro human proteome.

Matsumoto M. et al.: Nat Methods. 2017 Mar;14(3):251-258.

PMID: 28267743

・Expression of > 18,000 human recombinant proteins from AIST and ORFeome clone sets

・Digestion and mTRAQ△4 labeling

・MRM assay with mass tag (mTRAQ) labeled peptides

・Genome-wide peptide resource: database comprised 216,476 unique peptides for 17,973 proteins, corresponding to 86.3% of all (20,819) human protein-coding genes


High-throughput production of a stable isotope-labeled peptide library for targeted proteomics using a wheat germ cell-free synthesis system.

Takemori N. et al.: Mol Biosyst. 2016 July 19;12(8):2389-93.

PMID: 27203355

・High-throughput production of reference peptide library from 2201 selected peptides

・Designed 162 His-tagged QconCATs with on average 14 peptides

・Incorporation of 13C/15N labeled Lys and Arg during cell-free protein expression (up to 99% labeling efficiency for incorporating [13C, 15N]-L-Lys and [13C, 15N]-L-Arg)

・Gel-separated QconCATs were digested with trypsin

・71% of all peptides within the QconCATS identified during MS/MS analysis


MEERCAT: Multiplexed Efficient Cell Free Expression of Recombinant QconCATs For Large Scale Absolute Proteome Quantification.

Takemori N. et al.: Mol Cell Proteomics. 2017 Dec;16(12):2169-2183.

PMID: 29055021 PMCID: PMC5724179

・Use of wheat germ system to prepare QconCATs, artificial proteins comprising concatemers of multiple standard peptides, on high throughput for use in proteomics studies


A practical guide to the FLEXIQuant method.

Singh S. et al.: Methods Mol Biol. 2012;893:295-319.

PMID: 22665308

・Detailed protocol for using FLEXIQuant method for indirect protein quantification

・Isotope-labeled FLEX-tag quantified by reference to peptide

・Because of indirect quantification using FLEX-tag, no need for highly purified protein standards

・Commonly multiple peptides per target protein for better quantification/coverage

・FLEXIQuant method used in medical research

・The related FLEXIKinase method is using FLEXIQuant to study protein modification (PTM)


FLEXIQinase, a mass spectrometry-based assay, to unveil multikinase mechanisms.

Singh S.A. et al.: Nat Methods. 2012 Apr 8;9(5):504-8.

PMID: 22484849 PMCID: PMC3595540

・Mass spectrometry-based method that provides residue-resolved quantitative information about protein phosphorylation

・Combination of full-length stable isotope-labeled protein (FLEXIQuant) with traditional kinase assay to determine multikinase substrate phosphorylation

・Examples c-Jun N-terminal kinase (JNK)-dependent glycogen synthase kinase 3β (GSK3β) activity


FLEXITau: Quantifying Post-translational Modifications of Tau Protein in Vitro and in Human Disease.

Mair W. et al.: Anal Chem. 2016 Apr 5;88(7):3704-14.

PMID: 26877193 PMCID: PMC5808556

・Use of FLEXIQuant to study phosphorylation of the Tau protein related to Alzheimer's disease (AD)

・FLEXITau is used to measure phosphorylation stoichiometry obtaining an unbiased quantitative view of the tau post-translational modification (PTM)

・FLEXITau furhter defined the tau PTM landscape in AD post-mortem brain


*We supports FLEXIQuant by providing dedicated reagents, expression vector, and the reference peptide.

 Click here for more information.

Working with Biotinylated Proteins

The very strong non-covalent binding of biotin to avidin and streptavidin has been widely used in analytical assays, detection, and protein purification. Adding the biotin ligase BirA from E. coli to wheat germ cell-free protein expression reactions allows for specific mono-biotinylation of proteins having a short recognition sequence for the BirA ligase:


Simple screening method for autoantigen proteins using the N-terminal biotinylated protein library produced by wheat cell-free synthesis.

Matsuoka K. et al..: Proteome Res. 2010 Aug 6;9(8):4264-73.

PMID: 20575507 PMCID: PMC2917173

・Direct preparation of biotinylated proteins by adding BirA to translation reaction

・Development of HTP screening assay on PerkinElmer AlphaScreen™ (Amplified Luminescent Proximity Homogeneous Assay)

・Background free detection without need of protein purification

・Use of biotinylated proteins to screen serum samples for autoimmune antibodies


Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.

Tsuge S. et al.: J Histochem Cytochem. 2011 Jul;59(7):673-89.

PMID: 21525188 PMCID: PMC3201162

・Biotinylated protein used in different detection assays

・Biotinylated protein used in PerkinElmer AlphaScreen™ experiments (see above)


Profiling of autoantibodies in sera of pancreatic cancer patients.

Nagayoshi Y. et al.: Ann Surg Oncol. 2014 Jun;21 Suppl 3:S459-65.

PMID: 24585405

・Preparation of biotinylated protein library covering 2,183 human genes

・Screening of serum antibodies by PerkinElmer AlphaScreen™ method (see above)

・Identified potential biomarkers for pancreas cancer


Identification of new abscisic acid receptor agonists using a wheat cell-free based drug screening system.

Nemoto K. et al.: Sci Rep. 2018 Mar 9;8(1):4268.

PMID: 29523814 PMCID: PMC5844987

・Abscisic acid (ABA) is main phytohormone involved plant stress response

・Using wheat germ system and PerkinElmer AlphaScreen™ method in a drug screening system for biochemical validation ABA receptors

・Successful use of the system for screening compound library to identify ABA receptor agonists that are candidate agrichemicals

Ubiquitin E3 Ligases and DUB Proteins

The wheat germ cell-free protein expression system has been used very successfully in several studies on protein ubiquitination and E3 ubiquitin ligases:


Wheat germ-based protein libraries for the functional characterisation of the Arabidopsis E2 ubiquitin conjugating enzymes and the RING-type E3 ubiquitin ligase enzymes.

Ramadan A. et al.: BMC Plant Biol. 2015 Nov 10;15:275.

PMID: 26556605 PMCID: PMC4641371

・Use of RIKEN Arabidopsis full-length cDNA library in combination with template preparation by PCR to make protein libraries of Arabidopsis E2 and RING E3 enzymes

・In total 35 E2s and 204 RING proteins from Arabidopsis made for functional characterization

・Thioester assays using dithiothreitol (DTT) showed DTT-sensitive ubiquitin thioester formation for all E2s expressed

・All the 27 RING E3s tested showed ubiquitin ligase activity


Establishment of a Wheat Cell-Free Synthesized Protein Array Containing 250 Human and Mouse E3 Ubiquitin Ligases to Identify Novel Interaction between E3 Ligases and Substrate Proteins.

Takahashi H. et al.: PLoS One. 2016 Jun 1;11(6):e0156718.

PMID: 27249653 PMCID: PMC4889105

・Preparation of protein array containing 227 human and 23 mouse recombinant E3 ligases

・Using PerkinElmer AlphaScreenTM method (see above) for high-throughput binding assay

・Using MDM2 and p53 as target proteins for E3 ligases


The E3 ubiquitin ligase MIB2 enhances inflammation by degrading the deubiquitinating enzyme CYLD.

Uematsu A. et al.: Nat Commun. J Biol Chem. 2019 Sep 20;294(38):14135-14148.

PMID: 31366726 PMCID: PMC6755803

・Study uses wheat germ cell-free expression together with AlphaScreen™ assays to detect protein-protein interactions

・Results suggest that MIB2 enhances NF-κB signaling in inflammation by promoting the ubiquitin-dependent degradation of tumor suppressor CYLD


The ubiquitin ligase STUB1 regulates stability and activity of RUNX1 and RUNX1-RUNX1T1.

Yonezawa T. et al.: J Biol Chem. 2017 Jul 28;292(30):12528-12541

PMID: 28536267 PMCID: PMC5535027

・Preparation of 287 E3s in wheat germ cell-free expression system

・Identification of several RUNX1-interacting E3 ubiquitin ligases

・Out of those, STUB1 bound to RUNX1 and induced its ubiquitination and degradation mainly in the nucleus


Ubiquitin-proteasome system controls ciliogenesis at the initial step of axoneme extension.

Kasahara K. et al.: Nat Commun. 2014 Oct 1;5:5081.

PMID: 25270598 PMCID: PMC4205846

・Using a two-stepped global E3 screening process

・1,172 E3 ligase proteins (including putative E3s) were prepared from the human proteome expression resource library (HuPEX) using wheat germ cell-free expression system

・E3 ligase proteins involve in trichoplein polyubiquitylation and ciliogenesis were screened using a special protein array platform (“Protein Active Array”)



In addition to the expression and screening of E3 ligases, the wheat germ system was also used to prepare a comprehensive set of active human deubiquitinating enzymes (DUBs):


A Human DUB Protein Array for Clarification of Linkage Specificity of Polyubiquitin Chain and Application to Evaluation of Its Inhibitors.

Takahashi A. et al.: Biomedicines. 2020 Jun 4;8(6):152.

PMID: 32512835 PMCID: PMC7344921

・88 full-length recombinant human DUB proteins prepared

・80 showed DUB activities, and their linkage specificities were determined

・DUB screening assay based on AlphaScreenTM technology

Membrane Proteins


Membrane proteins are very difficult to handle, and their expression in many expression systems had failed due to interactions with the cell host. The problems can be avoided by using cell-free protein expression, where special reaction formats are available to conduct protein expression in the presence of detergents, nanodiscs, or add lipid vesicles to directly prepare proteoliposomes. CFS supports the expression of membrane proteins in our system by offering ready-to-use lyophilized liposomes in combination with the BD reaction format:


Production of monoclonal antibodies against GPCR using cell-free synthesized GPCR antigen and biotinylated liposome-based interaction assay.

Takeda H. et al.: Sci Rep. 2015 Jun 10;5:11333.

PMID: 26061673 PMCID: PMC4462149

・Expression of GPCRs in the presence of liposomes

・New BD protein expression reaction format combining bilayer with dialysis method

・Direct use of proteoliposomes in antibody production


Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

Nozawa A. et al.: BMC Biotechnol. 2011 Apr 11;11:35.

PMID: 21481249 PMCID: PMC3090341

・Used 40 mammalian membrane proteins having one to 14 transmembrane domains and five soluble proteins to test expression of membrane proteins in wheat germ cell-free expression system

・Their results indicate that the wheat cell-free system is a highly productive method for proteoliposome formation: At least 29 of the membrane proteins, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation


A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

Minkoff B.B.: J Biol Chem. 2017 Apr 7;292(14):5932-5942.

PMID: 28235802 PMCID: PMC5392584

・Plant receptor-like kinases RLK FERONIA is a peptide receptor

・Cell-free wheat germ expression system was used to co-translate mRNA encoding FERONIA mRNA together with mRNA encoding membrane scaffold protein variant MSP1D1

・The addition of the lipid cardiolipin allowed for assembly of both proteins into nanodiscs

・FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases


Modifications of wheat germ cell-free system for functional proteomics of plant membrane proteins.

Nozawa A., Tozawa Y.: Methods Mol Biol. 2014;1072:259-72.

PMID: 24136528

・They describe three methods for membrane protein production utilizing a wheat germ cell-free protein expression system

・Supplementation of liposomes or detergents allows the synthesis of functional integral membrane proteins

・Supplementation of myristic acid enables synthesis of N-myristylated peripheral membrane proteins


Wheat Germ Cell-Free Overexpression for the Production of Membrane Proteins.

Fogeron M.L. et al.: Methods Mol Biol. 2017;1635:91-108.

PMID: 28755365

・Method to produce a viral integral membrane protein for structural studies by solid-state NMR in a native-like lipid environment


Functional G-Protein-Coupled Receptor (GPCR) Synthesis: The Pharmacological Analysis of Human Histamine H1 Receptor (HRH1) Synthesized by a Wheat Germ Cell-Free Protein Synthesis System Combined with Asolectin Glycerosomes.

Suzuki Y. et al.: Front Pharmacol. 2018 Feb 6;9:38.

PMID: 29467651 PMCID: PMC5808195

・Glycerosomes (liposomes containing high concentrations of glycerol) have greater morphological stability than liposomes

・Use of glycerosomes instead of regular liposomes to express two GPCRs into proteoglycerosomes using the wheat germ system

・Functional analysis of expressed GPCRs to show feasibility for working with proteoglycerosomes


Engineered membrane protein antigens successfully induce antibodies against extracellular regions of claudin-5.

Hashimoto Y. et al.: Sci Rep. 2018 May 30;8(1):8383.

PMID: 29849184

・Approach to make antibodies against the extracellular regions (ECR) of membrane proteins

・Normalizing mRNA GC content improved expression of CLDN-5 protein in the cell-free system

・Antigen synthesized as proteoliposomes

・Successful monoclonal antibody developed targeting difficult-to-produce membrane proteins


Characterization of mitochondrial carrier proteins of malaria parasite Plasmodium falciparum based on in vitro translation and reconstitution.

Nozawa A. et al.: Parasitol Int. 2020 Jun 20;79:102160.

PMID: 32574727

・Expression of mitochondrial carrier (MC) membrane transporters form malaria parasite Plasmodium falciparum and Saccharomyces cerevisiae

・Functional properties of reconstituted MCs could reflect lipid content of their native membranes, where some P. falciparum proteins showed cardiolipin-dependent transport activities

Protein Structure and Folding

The wheat germ cell-free protein expression system has been used in different projects to determine information on protein folding and protein structures including the Protein Structural Initiative (PSI) in the US. In particular, labeled proteins for NMR studies have been made successfully using the open nature of a cell-free expression reaction. CFS provides amino acid free versions of our expression system to prepare labeled proteins for use in NMR:


A wheat germ cell-free system is a novel way to screen protein folding and function.

Morita E.H.: Protein Sci. 2003 Jun;12(6):1216-21.

PMID: 12761392 PMCID: PMC2323893

・Expression of two (15)N-labeled proteins in E. coli and wheat germ cell-free protein expression system

・Obtained (1)H-(15)N HSQC spectra from those proteins to obtain structural information

・Comparing the spectra, they showed that proteins synthesized with a wheat germ cell-free system have the proper protein folding and enough biological activity


Cell-free protein synthesis for functional and structural studies.

Makino S. et al.: Methods Mol Biol. 2014;1091:161-78.

PMID: 24203331

・Overview based on the experience learned using the wheat germ cell-free expression system during the Protein Structure Initiative (PSI) project for high-throughput protein production


Expression platforms for producing eukaryotic proteins: a comparison of E. coli cell-based and wheat germ cell-free synthesis, affinity and solubility tags, and cloning strategies.

Aceti D.J. et al.: J Struct Funct Genomics. 2015 Jun;16(2):67-80.

PMID: 25854603 PMCID: PMC4430420

・Use of 21 well-characterized eukaryotic proteins used as controls within the context of a structural genomics pipeline

・Steps included cloning, small-scale expression trials, large-scale growth or synthesis, and purification

・Successfully purified proteins were either crystallized or used in (1)H-(15)N HSQC NMR analyses


Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies.

Noirot C. et al.: N Biotechnol. 2011 Apr 30;28(3):232-8.

PMID: 20609396

・Use of wheat germ cell-free expression to produce recombinant proteins for solid-state NMR studies

・Productions of the prions Ure2p and HET-s for structural studies


NMR assignment method for amide signals with cell-free protein synthesis system.

Kohno T.: Methods Mol Biol. 2010;607:113-26.

PMID: 20204853

・They describe a method to produce dual amino acid-selective (13)C-(15)N labeled proteins for NMR using wheat germ cell-free expression system

・Method enables sequence-specific assignments of amide signals even for very large proteins


Cell-free protein production and labeling protocol for NMR-based structural proteomics.

Vinarov D.A. et al.: Nat Methods. 2004 Nov;1(2):149-53. Epub 2004 Oct 21.

PMID: 15782178

・They describe a wheat germ cell-free platform for protein production to support efficient NMR structural studies on eukaryotic proteins

・Expression of At3g01050.1 from Arabidopsis thaliana using a semicontinuous cell-free translation reaction to incorporate (15)N-labeled or (13)C, (15)N-labeled amino acids

・They obtained three-dimensional (3D) structure of At3g01050.1 showing that this protein is an unusual member of the beta-grasp protein family


Structural Studies of Self-Assembled Subviral Particles: Combining Cell-Free Expression with 110 kHz MAS NMR Spectroscopy.

David G. et al.: Angew Chem Int Ed Engl. 2018 Apr 16;57(17):4787-4791

PMID: 29457857

・Made milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) in wheat germ system

・Protein used in NMR structural analysis


Protein sample preparation for solid-state NMR investigations.

Lacabanne D. et al.: Prog Nucl Magn Reson Spectrosc. 2019 Feb 110:20-33.

PMID: 30803692

・Review on aspects of solid-state NMR sample preparation

・Describes use of wheat germ cell-free expression system


Protein structural biology using cell-free platform from wheat germ.

Novikova I.V. et al.: Adv Struct Chem Imaging. 2018;4(1):13.

PMID: 30524935 PMCID: PMC6244559

・They are using a wheat germ cell-free expression platform for obtaining functional proteins for structural biology

・Very good advice on template testing and scaling protein expression

・First example for using the wheat germ cell-free expression system for cryo-EM

Working with Genomic Resources

The wheat germ cell-free expression system has been used to study cDNA clones derived from large genomic resources. These applications have often used large clone numbers, and PCR methods for direct template preparation. In some cases, proteins were later used for protein array preparation. CFS can provide protocols to prepare expression templates by PCR:


Functional genomics using RIKEN Arabidopsis thaliana full-length cDNAs.

Seki M., Shinozaki K.: J Plant Res. 2009 Jul;122(4):355-66.

PMID: 19412652

・Review on the use and analysis of the RIKEN Arabidopsis thaliana full-length cDNA collection including protein studies using wheat germ cell-free expression system


An integrated immunoproteomics and bioinformatics approach for the analysis of Schistosoma japonicum tegument proteins.

Chen J.H. et al.: J Proteomics. 2014 Feb 26;98:289-99.

PMID: 24448400

・They combined immunoproteomics and bioinformatics approaches to profile the tegument of the human blood fluke Schistosoma japonicum

・Full-length tegument proteins were cloned and expressed at high-throughput using wheat germ cell-free expression system

・They screened sera from S. japonicum-infected patients and normal subjects using protein arrays


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.

PMID: 19054851

・They generated 33,275 human Gateway entry clones for protein synthesis

・They expressed 13,364 human proteins using wheat germ cell-free expression system

・Functional assessment of 75 tested phosphatases showed biological activity for 58 (77%) proteins

・Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system

・They manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins


The wheat germ cell-free protein expression system can be fully automated as for example done in the CFS Protemist DTII instrument that can perform all reaction steps from RNA synthesis to protein purification. For large-scale production, the translation reaction can be automated for repeated reagent supply as done in the CFS Protemist XE instrument. Both CFS instruments have been successfully used in protein production as given in the following examples:


Automated cell-free protein production methods for structural studies.

Beebe E.T. et al.: Methods Mol Biol. 2014;1140:117-35.

PMID: 24590713

・Overview on using different CFS protein synthesizers during structural genomics project


Development of oligomannose-coated liposome-based nasal vaccine against human parainfluenza virus type 3.

Senchi K. et al.: Front Microbiol. 2013 Nov 26;4:346.

PMID: 24324462 PMCID: PMC3840497

・Example for using Protemist XE for large-scale protein production during vaccine development

Special Applications and Use of Additives

A cell-free protein expression system uses an open in vitro reaction format that allows for modifying the reaction conditions to better match with protein requirements. In the following, some examples are given where additives like for example detergents had been used during the translation reaction:


Cell-free protein synthesis of membrane (1,3)-β-d-glucan (curdlan) synthase: co-translational insertion in liposomes and reconstitution in nanodiscs.

Periasamy A. et al.: Biochim Biophys Acta. 2013 Feb;1828(2):743-57.

PMID: 23063656

・The publication offers detailed information on the use of detergents and lipids in wheat germ cell-free expression reactions


Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

Genji T., Nozawa A., Tozawa Y.: Biochem Biophys Res Commun. 2010 Oct 1;400(4):638-42.

PMID: 20807510

・Example for use of detergents in translation reaction


Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.

Goren M.A. and Fox B.G.: Protein Expr Purif. 2008 Dec;62(2):171-8.

PMID: 18765284, PMCID: PMC2586813

・Expression of human stearoyl-CoA desaturase in the presence of unilamelar liposomes

・Co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes

・After in vitro reconstitution of non-heme iron and heme active sites, the function of reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA

・Publication provides information on element and lipid analysis of wheat germ extractPublication provides information on element and lipid analysis of wheat germ extract


Apoglobin Stability Is the Major Factor Governing both Cell-free and in Vivo Expression of Holomyoglobin.

Samuel P.P.: J Biol Chem. 2015 Sep 25;290(39):23479-95.

PMID: 26205820 PMCID: PMC4583012

・They used wheat germ cell-free protein expression system to examine quantitatively the factors that govern expression of holoMb

・Their results demonstrated that the cell-free transcription/translation system can be used as a high throughput platform to screen for apoglobin stability


The quiescin sulfhydryl oxidase (hQSOX1b) tunes the expression of resistin-like molecule alpha (RELM-α or mFIZZ1) in a wheat germ cell-free extract.

Gad W. et al.: PLoS One. 2013;8(1):e55621.

PMID: 23383248 PMCID: PMC3561318

・Example for expressing in the wheat germ cell-free expression system proteins having disulfide bonds by adding cofactor to expression reaction


Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins.

Gad W. et al.: Biosci Rep. 2014 Jul 29;34(4). pii: e00122.

PMID: 24924257 PMCID: PMC4114062

・They developed a wheat germ cell-free expression system for the expression of the highly toxic Aah (Androctonus australis hector) II protein that requires the proper formation of four disulfide bonds

・Soluble, recombinant GST-tagged AahII toxin was obtained, and the purified rAahII was highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice

・An LD50 (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicated that the wheat germ cell-free expression system produces properly folded and biological active rAahII


Functional expression, purification, characterization, and membrane reconstitution of non-structural protein 2 from hepatitis C virus.

Fogeron M.L. et al.: Protein Expr Purif. 2015 Dec;116:1-6.

PMID: 26325423

・Non-structural protein 2 (NS2) of hepatitis C virus (HCV) is an integral membrane protein that contains a cysteine protease

・Using a wheat germ cell-free expression system, they produced and purify milligram amounts of a detergent-solubilized full-length NS2 exhibiting the expected secondary structure features


Wheat germ cell-free expression: Two detergents with a low critical micelle concentration allow for production of soluble HCV membrane proteins.

Fogeron M.L. et al.: Protein Expr Purif. 2015 Jan;105:39-46.

PMID: 25306874

・Use of wheat germ cell-free protein expression system in the presence of various detergents to produce the non-structural membrane proteins 2, 4B and 5A of the hepatitis C virus (HCV)

・They showed that lauryl maltose neopentyl glycol (MNG-3) and dodecyl octaethylene glycol ether (C12E8) detergents can yield essentially soluble membrane proteins at detergent concentrations that do not inhibit the cell-free protein expression reaction


Rational optimization of amber suppressor tRNAs toward efficient incorporation of a non-natural amino acid into protein in a eukaryotic wheat germ extract.

Ogawa A., Namba Y., Gakumasawa M.: Org Biomol Chem. 2016 Mar 7;14(9):2671-8.

PMID: 26832824

・Amber suppression can be used for genetically incorporating a non-natural amino acid (NAA) into a protein during translation by utilizing an NAA-charged amber suppressor tRNA (sup-tRNA)

・They optimized amber sup-tRNAs to efficiently incorporate a model NAA, p-acetyl-phenylalanine (AcPhe), into a protein using the wheat germ cell-free protein expression system


Wheat germ cell-free expression system as a pathway to improve protein yield and solubility for the SSGCID pipeline.

Guild K. et al.: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2011 Sep 1;67(Pt 9):1027-31.

PMID: 21904045 PMCID: PMC3169397

・The SSGCID program used wheat germ cell-free protein expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli

・Testing indicates that the system is a valuable tool for these protein targets

・Protein solubility could be increased by the addition of the NVoy polymer to the reaction mixture

・These data indicate that wheat germ cell-free protein expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein


Extent and Origins of Functional Diversity in a Subfamily of Glycoside Hydrolases.

Glasgow E.M. et al.: J Mol Biol. 2019 Mar 15;431(6):1217-1233.

PMID: 30685401

・Template preparation by gene synthesis followed by expression in wheat germ system

・Glycoside hydrolase family 5 members were screened for activity of the catalytic core domains from subfamily 4 (GH5_4) and closely related enzymes on four substrates: lichenan, xylan, mannan, and xyloglucan

・Discussing several possibilities for the ongoing evolutionary specialization of GH5_4 enzymes


Preparation of a Millimeter-Sized Supergiant Liposome That Allows for Efficient, Eukaryotic Cell-Free Translation in the Interior by Spontaneous Emulsion Transfer.

Takahashi H. and Ogawa A.: ACS Synth Biol. 2020 Jul 17;9(7):1608-1614.

PMID: 32559381

・Preparation millimeter-sized supergiant unilamellar vesicles (SGUVs) by spontaneous emulsion transfer to encapsulate cell-free protein expression system

・Encapsulated wheat germ translation system allowed for protein synthesis with a high efficiency comparable to that outside a liposome

Use in Malaria Research

The wheat germ cell-free protein expression system proved very powerful to express malaria proteins for biochemical studies. The following publications give some examples on its use in malaria research:


Leveraging the wheat germ cell-free protein synthesis system to accelerate malaria vaccine development. (review article)

Kanoi BN et al.: Parasitol Int. 2020 Oct 30;80:102224.

PMID: 33137499

・The wheat germ cell-free protein synthesis system has a record for identifying and evaluating targets for malaria vaccine development

・Progresses for novel antigen discovery benefit from the throughput and scalability of the wheat germ system serving screening and evaluation steps in serology and immunization tests


Malaria transmission-blocking vaccines: wheat germ cell-free technology can accelerate vaccine development. (review article)

Miura K. et al.: Expert Rev Vaccines. 2019 Oct;18(10):1017-1027.

PMID: 31566026

・Review on progress and prospect of malaria transmission blocking vaccines (TBV) research and development

・Applying wheat germ cell-free protein synthesis to accelerate TBV development


Identification of domains within Pfs230 that elicit transmission blocking antibody responses.

Tachibana M. et al.: Vaccine. 2019 Mar 22;37(13):1799-1806.

PMID: 30824357 PMCID: PMC6708081

・Pfs230 is a promising candidate to develop a transmission blocking vaccine for P. falciparum malaria

・Expression of protein fragments to identify transmission blocking domain

・Protein fragments used for antibody production and later analysis

・Antibodies against protein fragments including a CM domain 1 showed transmission blocking activity


Discovery of Novel Plasmodium falciparum Pre-Erythrocytic Antigens for Vaccine Development.

Aguiar J.C. et al.: PLoS One. 2015 Aug 20;10(8):e0136109.

PMID: 26292257 PMCID: PMC4546230

・Selection of 27 P. falciparum pre-erythrocytic antigens that were then expressed in the wheat germ system

・Tested the protein of sera from humans immunized with P. falciparum radiation-attenuated sporozoites (RAS)

・Their results provided evidence to further evaluate these antigens as vaccine candidates


Naturally acquired antibody responses to more than 300 Plasmodium vivax proteins in three geographic regions.

Longley R.J. et al.: PLoS Negl Trop Dis. 2017 Sep 11;11(9):e0005888.

PMID: 28892517 PMCID: PMC5614652

・hey selected 307 P. vivax proteins that had been prepared in the wheat germ system

・They measured antibody responses against those 307 P. vivax proteins at the time of P. vivax infection and at 2-3 later time-points in three countries

・The data suggest that IgG seropositivity rates including magnitude and longevity are features that are relate to the individual proteins


Identification of highly-protective combinations of Plasmodium vivax recombinant proteins for vaccine development.

França C.T. et al.: Elife. 2017 Sep 26;6. pii: e28673.

PMID: 28949293 PMCID: PMC5655538

・They measured total IgG antibodies to 38 P. vivax antigens expressed in different systems including wheat germ to investigate the prospective risk of malaria

・Obtained valuable data to establish a clear path forward to testing a multicomponent P. vivax vaccine


Antibody profiles to wheat germ cell-free system synthesized Plasmodium falciparum proteins correlate with protection from symptomatic malaria in Uganda.

Kanoi B.N. et al.: Vaccine. 2017 Feb 7;35(6):873-881.

PMID: 28089547

・They prepared a library of 1,827 P. falciparum proteins derived from 1565 genes representing 30% of the entire P. falciparum genome

・Proteins were expressed in the wheat germ system

・Immunoreactivity to patient sera was determined by the PerkinElmer AlphaScreenTM method (see above)

・Down selected to 53 uncharacterized proteins not previously characterized as vaccine candidates


Immunoscreening of Plasmodium falciparum proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate.

Morita M. et al.: Sci Rep. 2017 Apr 5;7:46086.

PMID: 28378857 PMCID: PMC5380959

・Use of same library of 1,827 P. falciparum proteins made in the wheat germ system

・Screened purified IgGs from residents in malaria endemic area to identify antibodies against malaria proteins

・They identified LSA3 as a novel blood-stage vaccine candidate


We are actively contributed to projects for identifying vaccine candidates and biomarkers for malaria elimination supported the Global Health Innovative Technology (GHIT) Fund of Japan. You can find more information on those projects on our homepage at here.


Please visit the homepage from GHIT to learn more about their important work and supported projects.

Preparation and Use of Human Protein Bead Array


CFS uses the wheat germ cell-free protein expression system to prepare our human protein bead array. We selected this format to keep proteins always in solution during the entire experiment and to avoid protein denaturation. This array holds ~20,000 human full-length proteins and can be used for studying protein-protein interactions. In the following example it had been used to characterize antibodies for their binding specificity:


CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins

Morishita R. et al.: Sci Rep. 2019 Dec 18;9(1):19349.

PMID: 31852950 PMCID: PMC6920144 

・Presenting ~20,000 proteins on 1536-well plate with up to 14 proteins per well

・Use plate with multiple targets to screen antibodies for binding to human proteins

・Re-array proteins from positive wells to identify individual antibody targets

・Mapping of binding sites combining protein alignments with mutation screeningMapping of binding sites combining protein alignments with mutation screening

Enzyme Discovery and Engineering

The wheat germ cell-free protein expression system has been used in enzyme discovering to improve for example biofuel production and to develop a new biotinylation enzyme for protein-protein interaction analysis:


Cell-free translation of biofuel enzymes.

Takasuka T.E. et al.: Methods Mol Biol. 2014;1118:71-95.

PMID: 24395410 PMCID: PMC5820533

・Use of Protemist DTII to screen for new enzymatic activities


AirID, a novel proximity biotinylation enzyme, for analysis of protein-protein interactions.

Kido K. et al.: Elife. 2020 May 11;9:e54983.

PMID: 32391793, PMCID: PMC7302878

・Reconstitution of ancestral BirA enzyme from metagenomic data

・Enzymatic characterization of newly designed BirA enzymes

・Biochemical characterization of AirID enzyme

Biomarker Discovery, Viral Proteins, and Development of Diagnostic Assays

The wheat germ cell-free protein expression system is a valuable tool to prepare proteins for the discovery of new biomarkers, antibody validation, and rapid development of diagnostic assays. Additional examples for the expression of viral proteins are already listed above:


Development and validation of serological markers for detecting recent Plasmodium vivax infection.

Longley R.J.; White M.T. et al.: Nat Med. 2020 May;26(5):741-749.

PMID: 32405064

・Screening for markers to identify patients with recent P. vivax malaria infection

・Marker set for serological screening of patients developed which could be used for screen and treat companies to prevent relapses of the disease


In vitro translation of virally-encoded replication polyproteins to recapitulate polyprotein maturation processes.

Habersetzer J. et al.: Protein Expr Purif. 2020 Jul 15;105694.

PMID: 32681958

・Expression of viral polyproteins from human norovirus and plant tymovirus

・Proteins had fully functional active protease domain and underwent spontaneous auto-cleavage

・Introduction of mutation to block proteolytic maturationIntroduction of mutation to block proteolytic maturation


Whole nucleocapsid protein of SARS-CoV-2 may cause false positive results in serological assays.

Yamaoka Y. et al.: Clin Infect Dis. 2020 May 23;ciaa637.

PMID: 32445559, PMCID: PMC7314131

・Expression of nucleocapsid protein (NP) of SARS-CoV-2 as full-length protein and with an N-terminal deletion

・Proteins used in development of commercial SARS-CoV-2 tests


A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors.

Matsunaga S. et al.: Front Microbiol. 2015 Oct 31;6:1220.

PMID: 26583013 PMCID: PMC4628118

・Novel in vitro method for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR)

・Using wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure

・Enzymatic activity of PRs measured by AlphaScreenTM in the presence or absence of clinically used protease inhibitors


Development of Monoclonal Antibody and Diagnostic Test for Middle East Respiratory Syndrome Coronavirus Using Cell-Free Synthesized Nucleocapsid Antigen.

Yamaoka Y. et al.: Front Microbiol. 2016 Apr 20;7:509.

PMID: 27148198, PMCID: PMC4837155

・Production of MERS-CoV NP antigen

・Production of monoclonal antibodies for use in commercial MERS-CoV test

Studying Protein-Protein Interactions

Easy access to modified proteins using a cell-free expression system also provides a great tool to better study protein-protein interactions on different platforms including the PerkinElmer AlphaScreen™ technology or Surface Plasmon Resonance (SPR) that can use biotinylated proteins as mentioned above and for example combines them with tagged proteins to confirm binding of the partners. Protein-protein interactions have been studied with the wheat germ system in plant, animal, and viral research:


Viral Research

Involvement of the 3' Untranslated Region in Encapsidation of the Hepatitis C Virus.

Shi G. et al.: PLoS Pathog. 2016 Feb 11;12(2):e1005441.

PMID: 26867128 PMCID: PMC4750987

・Study on selective packaging of the HCV genome into viral particles

・Identification of 3' UTR element that acts in cis for encapsidation of viral genome

・Using wheat germ system to express FLAG-tagged HCV Core and its mutants to perform RNA binding assays with biotinylated RNA on AlphaScreen™


HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains.

Shibata Y. et al.: PLoS Pathog. 2017 Jan 19;13(1):e1006162.

PMID: 28103322 PMCID: PMC5283754

・Study on Tax protein of human T-cell leukemia virus type 1 (HTLV-1)

・Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex

・Data suggest that Tax could lead to trans-autophosphorylation-mediated IKK activation

・GST, GST-HOIL-1L, GST-HOIP and GST-Sharpin were prepared by wheat germ cell-free protein synthesis during this study


PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

Miyakawa K. et al.: Nat Commun. 2019 Apr 23;10(1):1844.

PMID: 31015445 PMCID: PMC6479052

・PIM family of serine/threonine protein kinases phosphorylate Vpx protein

・Using proteomics and functional analysis to show that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions

・Initial screen used a set of 412 human protein kinases



Plant Research

Members of the Plant CRK Superfamily Are Capable of Trans- and Autophosphorylation of Tyrosine Residues.

Nemoto K. et al.: J Biol Chem. 2015 Jul 3;290(27):16665-77

PMID: 25969537 PMCID: PMC4505418

・Arabidopsis calcium-dependent protein kinase (CDPK/CPK)-related PKs (CRKs) have high Tyr-autophosphorylation activity and can phosphorylate Tyr residue(s) on substrate proteins in Arabidopsis

・Examined autophosphorylation activity of 759 protein kinases using an Arabidopsis protein array and wheat cell-free expression system

・Substrate screening for CRK3 by pulldown assay using protein expressed in wheat cell-free system and protein extracts prepared from cultured Arabidopsis cells

・Found Tyr-autophosphorylation activity for 38 protein kinases included into the screen


Tyrosine phosphorylation of the GARU E3 ubiquitin ligase promotes gibberellin signalling by preventing GID1 degradation.

Nemoto K. et al.: Nat Commun. 2017 Oct 17;8(1):1004.

PMID: 29042542 PMCID: PMC5645313

・Gibberellin (GA) is a major hormone for plant growth and development

・Study shows that GARU (GA receptor RING E3 ubiquitin ligase) mediates ubiquitin-dependent degradation of GID1

・TAGK2 plant Tyr-kinase is a target of genistein and inhibits GARU-GID1A interactions by phosphorylation of GARU at Tyr321

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions


OsMYC2, an essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that enhance its transactivation ability.

Ogawa S. et al.: Sci Rep. 2017 Jan 9;7:40175.

PMID: 28067270 PMCID: PMC5220304

・Jasmonic acid (JA) signaling induces basic helix-loop-helix transcriptional factor OsMYC2 and enhanced the activity of the OsNOMT promoter

・OsMYC2 interacts with OsMYL1 and OsMYL2 to further enhance transactivation activity of OsMYC2

・OsMYL1 and OsMYL2 support JA signaling via OsMYC2 and play role in production of sakuranetin in rice

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions



Animal Research

Bach2-Batf interactions control Th2-type immune response by regulating the IL-4 amplification loop.

Kuwahara M. et al.: Nat Commun. 2016 Sep 1;7:12596.

PMID: 27581382 PMCID: PMC5025763

・Study shows that Bach2 associates with Batf and binds to the regulatory regions of the Th2 cytokine gene loci

・This study used the AlphaScreen™ system with proteins made in the wheat germ system to study protein–protein interactions

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