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One system to express them all

PSI-SGKB [doi:10.1038/th_psisgkb.2009.50]
Technical Highlight - November 2009
Short description: A fast and effective protein-expression system that works for any organism could revolutionize protein production.

Tagging a gene of interest with a species-independent translation sequence (SITS) enables cell-free synthesis of the gene product. The method works with cell extracts of the protozoan Leishmania tarantolae, of the bacterium Escherichia coli and of four eukaryotes, suggesting that it is applicable to a broad variety of organisms.

Reliable protein expression systems are essential, whether you're working on a structural genomics project or on an individual study. Not only do you need to express a protein, you need it to have the same biological activity as the native protein, whatever the species. A recently published cell-free expression system that works for any species looks to be the way forward 1 .

Expression in Escherichia coli is the first method that most labs try, but it is not necessarily the best system for correctly expressing all eukaryotic proteins. This is partly because it lacks the chaperones required for folding complex eukaryotic proteins and is not able to perform specific eukaryotic post-translational modifications.

One way of expressing eukaryotic proteins is to use a eukaryotic cell-free expression system, such as that based on wheat-germ extract, which is PSI CESG's approach to producing proteins for crystallography. This particular system gives improved protein solubility, but the components are more expensive and more difficult to get hold of than the E. coli system. A universal cell-free translation set-up that could be used for any eukaryote from protozoa to humans would be highly desirable.

Initiating translation is usually the most difficult step in cell-free translation, often due to secondary structure in the mRNA which requires specialized mechanisms to overcome it. To get round this, Mureev et al. developed universal species-independent translational sequences (SITS) to replace the untranslated 5′ regions (UTRs) in mRNAs that are recognized by the translation initiation machinery.

The SITS are enriched with poly(A) sequences, which do not form secondary structure, and also contain unstable hairpin structures after the AUG initation codon to facilitate its recognition by the small ribosomal subunit. This approach avoids problems of secondary structure and other features of UTRs that might prevent mRNAs from being correctly recognized by the initiation machinery in vitro. SITS promote the assembly of the active ribosome–mRNA complexes that are necessary for translation, presumably without the need for initiation factors.

The team then developed a cell-free translation system based on extracts of the unicellular protozoan Leishmania tarentolae, which is easy and cheap to culture. In conjunction with SITS-containing PCR-transcribed RNAs, this system could produce 300 micrograms per milliliter of a recombinant cytoplasmic protein in 2 hours, and could translate mRNAs from cytoplasmic proteins from L. tarentolae itself, four other eukaryotes and even E. coli.

This set-up would also suit coexpression of different proteins and is likely to provide a straightforward way to study protein–protein and protein–small molecule interactions. It may well lead the way to high-throughput engineering of multi-protein complexes.

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References

  1. S. Mureev, O. Kovtun, U. Nguyen & K. Alexandrov Species-independent translational leaders facilitate cell-free expression.
    Nature Biotechnol. 27, 747-752 (2009). doi:10.1038/nbt.1556

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