MENU

Reference list | CellFree Sciences

header jp

header en

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

Overview

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

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)

 

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

Work on Ubiquitin E3 Ligases

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 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

 

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

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

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

Automation

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:

 

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

 

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

 

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

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:

 

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.

Copyright © CellFree Sciences Co.,Ltd. All Rights Reserved. [login]

We use cookies to ensure you get the best experience. By using our website you agree to our Cookie Policy.