Wheat Germ Cell‐Free Expression System for Protein Production

  title={Wheat Germ Cell‐Free Expression System for Protein Production},
  author={Dmitriy A. Vinarov and Carrie L. Loushin Newman and Ejan M. Tyler and John L. Markley and Mark N. Shahan},
  journal={Current Protocols in Protein Science},
The Center for Eukaryotic Structural Genomics, in cooperation with Ehime University and CellFree Sciences, has developed a novel wheat germ cell‐free technology for the production of eukaryotic proteins. Protein production and purification are robust and scalable for high‐throughput applications. The protocols have been used to express and purify proteins from Arabidopsis thaliana, human, mouse, rat and zebra fish. This unit describes expression and purification protocols for both small‐scale… 

Wheat germ cell‐free platform for eukaryotic protein production

A platform that utilizes wheat germ cell‐free technology to produce protein samples for NMR structure determinations is described and their application to targets chosen from a variety of eukaryotic genomes is described.

Cell-free protein synthesis technology in NMR high-throughput structure determination.

The integration of isotopically labeled proteins into the sequence of experiments required for NMR structure determination is outlined, and additional protocols for production of integral membrane proteins in the presence of either detergents or unilamellar liposomes are presented.

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

The tremendous potential of the rapidly evolving and highly versatile CFPS systems are highlighted, making them more widely used as common tools to recombinantly prepare particularly challenging recombinant eukaryotic proteins.

Trends in Wheat Germ Cell Free Protein Expression System with an Emphasis on Up-scaling and Industrial Application

This work compares the uses of different cell-free translation systems and reviews recent findings that support the possibility of scaling up and thereby enhancing the industrial application of wheat germ cell free protein expression.

The Center for Eukaryotic Structural Genomics

The technological innovations that underlie CESG’s platforms for bioinformatics and laboratory information management, target selection, protein production, and structure determination by X-ray crystallography or NMR spectroscopy are described.

Cell‐free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane

Cell‐free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives is reported, indicating that the procedure for cell‐ free synthesis produces protein structurally similar to that prepared from the cell membranes.

Cleavable C-terminal His-tag vectors for structure determination

A series of ligation-independent cloning expression vectors that append various cleavable C-terminal tags instead of the conventional N-Terminal tags are developed, and are cleavability with TEV or with both TEV and TVMV proteases.

Untranslated regions of diverse plant viral RNAs vary greatly in translation enhancement efficiency

BTEs confer the highest levels of translation of uncapped mRNAs in vitro and in vivo, while the capped omega sequence is most efficient in tobacco cells.



Comparison of cell‐based and cell‐free protocols for producing target proteins from the Arabidopsis thaliana genome for structural studies

In general, the wheat germ cell‐free platform offers the advantage of greater genome coverage for NMR‐based structural proteomics whereas the E. coli platform when successful yields more protein, as currently needed for crystallization trials for X‐ray structure determination.

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

A wheat germ cell-free platform for protein production that supports efficient NMR structural studies of eukaryotic proteins and offers advantages over cell-based methods is described.

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

This work has synthesized two 15N‐labeled proteins and obtained 1H‐15N HSQC spectra, and shown that proteins synthesized with a wheat germ cell‐free system have the proper protein folding and enough biological activity.

Protein expression systems for structural genomics and proteomics.

  • S. Yokoyama
  • Biology
    Current opinion in chemical biology
  • 2003

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

A cell-free system based on the eukaryotic translation apparatus of wheat seeds, which bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

Cell‐free production and stable‐isotope labeling of milligram quantities of proteins

Efficient synthesis of a disulfide-containing protein through a batch cell-free system from wheat germ.

Results show that the wheat germ cell-free system can produce a functional scFv with a simple change of the reaction ingredients, and it is shown that biotinylated scFV could be synthesized in similar fashion and immobilized on a solid surface to which streptavidin is bound.

A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes.

The results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable, contrasting with the common belief that cell-free translation systems are inherently unstable, even fragile.