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PSI-SGKB [doi:10.1038/th_psisgkb.2009.26]
Technical Highlight - June 2009
Short description: Wheat germ cell-free extract might be the answer to protein production difficulties.FEBS J. 273, 4160-4169 (2006)

Most laboratories use Escherichia coli to express and purify proteins from introduced genes, but this approach is often unsuccessful for eukaryotic proteins and can result in low or no expression. In vitro cell-free methods might provide an answer.

PSI CESG has developed a wheat germ cell-free protein production system in collaboration with Ehime University in Japan and the Japanese company CellFree Sciences 1 . They use a four-step approach to optimize protein production, starting with the creation of a suitable plasmid, small-scale screening (with 20–50 microliters) to monitor protein production and solubility, larger-scale (4–12 milliliters) production of labeled protein to test its suitability for structural studies and, finally, the production of larger quantities of protein.

A demonstration of PSI CESG's wheat germ cell-free translation system with green fluorescent protein messenger RNA.

The main advantages of the wheat germ cell-free method over E. coli are that more target proteins are produced as folded, soluble proteins, and it is also relatively simple to label proteins with isotopes for NMR spectroscopy or to add selenomethionine for X-ray crystallography. It is also handy for proteins that are toxic to cells, and for cutting out steps such as cell harvesting and lysis. Protein purification can often be simpler too, because the protein is more concentrated in the first place. The downside is that the wheat-germ system requires more expensive reagents than the E. coli system and these can be harder to get hold of.

As a further proof of the system's utility, Goren and Fox 2 have used it to produce a functional human integral membrane desaturase complex. So cell-free translation systems look promising for the expression and purification of many integral membrane proteins.

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References

  1. D. A. Vinarov, C. L. Loushin Newman and J. L. Markley Wheat germ cell-free platform for eukaryotic protein production.
    FEBS J. 273, 4160-4169 (2006). doi:10.1111/j.1742-4658.2006.05434.x

  2. M. A. Goren and B. G. Fox. Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex.
    Protein Expr. Purif. 62, 171-178 (2008). doi:10.1016/j.pep.2008.08.002

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