The objective of structural proteomics is to determine the structures of all protein folds found in nature and develop a public resource to organize and analyze protein structures and fold families. High throughput (HTP) methods, which can process multiple samples in parallel, saving both time and cost, play important roles in achieving this goal. Using C. elegans and human as model organisms, a HTP cloning and expression pipeline was developed for structural proteomics that required production of a large number of recombinant proteins, applying the Gateway cloning/expression technology and utilizing a stepwise automation strategy on an integrated robotic platform. This system can process up to 384 unique samples in parallel and successfully automates most aspects of gene cloning and protein expression analysis, from PCR to protein solubility profiling.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Service, R. F. (2001) Robots enter the race to analyze proteins. Science 292, 187–188.
Stevens, R. C., Wilson, I. A. (2001) Industrializing structural biology. Science 293, 519–520.
Hartley, J. L., Temple, G. F., and Brasch, M. A. (2000) DNA cloning using in vitro site-specific recombination. Genome Res. 10, 1788–1795.
Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S., and Vidal, M. (2000) GATEWAY recombinational cloning: Application to the cloning of large numbers of open reading frames or ORFeomes. Methods Enzymol. 328, 575–592.
Reboul, J., Vaglio, P., Rual, J.-F., Lamesch, P., Martinez, M., Armstrong, C. M., Li, S., Jacotot, L., Bertin, N., Janky, R., et al. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat. Genet. 34, 35–41.
Rual, J. F., Hirozane-Kishikawa, T., Hao, T., Bertin, N., Li, S., Dricot, A., Li, N., Rosenberg, J., Lamesch, P., Vidalain, P. O., Clingingsmith, T. R., Hartley, J. L., Esposito, D., Cheo, D., Moore, T., Simmons, B., Sequerra, R., Bosak, S., Doucette-Stamm, L., Le Peuch, C., Vandenhaute, J., Cusick, M. E., Albala, J. S., Hill, D. E., and Vidal M. (2004) Human ORFeome version 1.1: a platform for reverse proteom-ics. Genome Res. 14, 2128–2135.
Symersky, J., Zhang, Y., Schormann, N., Li, S., Bunzel, R., Pruett, P., Luan, C. H., and Luo, M. (2004) Structural genomics of Caenorhabditis elegans: structure of the BAG domain. Acta. Crystallogr. D. Biol. Crystallogr. 60, 1606–1610.
Lu, S., Symersky, J., Li, S., Carson, M., Chen, L., Meehan, E., and Luo, M. (2004) Structural genomics of Caenorhabditis elegans: crystal structure of the tropomodulin C-terminal domain. Proteins 56, 384–386.
Yoon, J., Kang, Y., Kim, K., Park, J., and Kim, Y. (2005) Identification and purification of a soluble region of BubR1: a critical component of the mitotic checkpoint complex. Protein Expr. Purif. 44, 1–9.
Finch, D., and Webb, M. (2005) Identification and purification of a soluble region in the breast cancer susceptibility protein BRCA2. Protein Expr. Purif. 40, 177–182.
Kagawa, N., Kemmochi, K., and Tanaka, S. (2004) One-step adapter PCR method for HTP Gateway technology cloning. Quest 1, 53–55.
Acknowledgments
This work was supported in part by the NIH grant 1P50-GM62407.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Chen, Y., Qiu, S., Luan, CH., Luo, M. (2008). A High Throughput Platform for Eukaryotic Genes. In: Kobe, B., Guss, M., Huber, T. (eds) Structural Proteomics. Methods in Molecular Biology™, vol 426. Humana Press. https://doi.org/10.1007/978-1-60327-058-8_13
Download citation
DOI: https://doi.org/10.1007/978-1-60327-058-8_13
Publisher Name: Humana Press
Print ISBN: 978-1-58829-809-6
Online ISBN: 978-1-60327-058-8
eBook Packages: Springer Protocols