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  • Applications of the PURE System

    Protein ExpressionReturn to Cell-Free Expression

    The advantages of the PURE system have been demonstrated in various in vitro applications including:

    1. High throughput functional genomics and proteomics

      The simple format of the PURE system allows it to be easily integrated into high throughput platforms for functional genomics and proteomics studies. The absence of any nuclease activities ensures the stability of linear DNA templates during protein synthesis. Individual DNA templates for in vitro expression can be generated by PCR, eliminating the time-consuming cloning process. This feature is particularly useful for high throughput screening at the whole genome scale, either for novel activities or for protein-protein interactions. For structural genomics projects, the PURE system can be an alternative route to acquire difficult protein targets which resist traditional cellular expression (2).

    2. Protein engineering

      Directed evolution of proteins in vitro is a powerful tool for improving and creating biocatalysts. A number of in vitro evolution methodologies, such as mRNA display (3), ribosome display (4) and in vitro compartmentalization (5), depend on in vitro translation. For example, NEB first demonstrated that the PURE system is uniquely suited for the in vitro selection of restriction endonucleases using the in vitro compartmentalization method (6), as it is free of nonspecific nuclease activity. In this study, the PURE system and the DNA library were dispersed into more than 109 aqueous droplets in a water-in-oil emulsion. The droplet encapsulation provides a linkage between the phenotype (expressed protein) and the genotype (DNA), which sets the stage for the specific selection of restriction enzyme genes. Other researchers have reported that using the PURE system greatly improves the efficiency of ribosome display (7). Systematically mutagenized protein-coding libraries can be used as well to test if a specific mutation(s) affects protein function. Again, only a few PCR steps are needed to obtain the mutant protein, providing a quick experimental verification of hypotheses.

    3. Study of protein expression, translation and folding

      The PURE system contains the minimal set of factors necessary for in vitro protein translation. It is largely free of chaperones and other cellular factors for post-translational modifications, thus providing a starting point to study the involvement of these factors in transcription/translation regulation and nascent chain folding (8). It can also be used to produce “clean” proteins which, if purified from traditional cellular hosts, may come with undesired modifications or bound co-factors. A number of research labs studying translation routinely use home-made reconstituted systems to study different aspects of translation.

    4. Incorporation of unnatural amino acids

      Another important advantage of the PURE system is the ability to control its composition. For example, omission of the release factor 1 (RF1) in the PURE system allows unnatural amino acids to be efficiently incorporated at specific amber codon sites via chemically mis-acylated suppressor tRNA (1,9). It was recently reported that the translation apparatus of E. coli can tolerate a wide range of amino acid derivatives, revealing even greater potential for the ribosomal synthesis of unnatural peptides using reconstituted systems (10).

    References

    1. Shimizu, Y., et al. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755. PMID: 11479568
    2. Graslund, S., et al. (2008) Nat. Methods, 5, 135–146. PMID: 18235434
    3. Roberts, R.W. and Szostak, J.W. (1997) Proc. Natl. Acad. Sci. USA, 94, 12297–12302. PMID: 9356443
    4. Hanes, J. and Pluckthun, A. (1997) Proc. Natl. Acad. Sci. USA, 94, 4937–4942. PMID: 9144168
    5. Tawfik, D.S. and Griffiths, A.D. (1998). Nat Biotechnol. 16, 652–656. PMID: 9661199
    6. Zheng, Y. and Roberts, R.J. (2007) Nucleic Acids Res. 35, e83. PMID: 17567609
    7. Villemagne, D., Jackson, R. and Douthwaite, J.A. (2006) J. Immunol. Methods, 313, 140–148. PMID: 16730021
    8. Kaiser, C.M., et al. (2006) Nature, 444, 455–460. PMID: 17051157
    9. Noren, C.J., et al. (1989) Science, 244, 182–188. PMID: 2649980
    10. Hartman, M.C., et al. (2007) PLoS ONE, 2, e972. PMID: 17912351

    PURExpress® is a registered trademark of New England Biolabs, Inc.