Approximately one third of the proteins encoded in prokaryotic and eukaryotic genomes reside in the membrane. However, membrane proteins comprise only a minute fraction of the entries in protein structural databases. This disparity is largely due to inherent difficulties in the expression and purification of sufficient quantities of membrane targets. To begin addressing the challenges of membrane protein production for high throughput structural proteomics efforts, the authors sought to develop a simple strategy that would permit the standardization of most procedures and the exploration of large numbers of proteins. Successful methods that have yielded membrane protein crystals suitable for structure determination were surveyed first. A number of recurrent trends in the expression, solubilization, purification, and crystallization techniques were identified. Based largely on these observations, a robust strategy was then developed that rapidly identifies highly expressed membrane protein targets and simplifies their production for structural studies. This method has been used to express and purify intramembrane proteases to levels sufficient for crystallization. This strategy is a paradigm for the purification of many other membrane proteins, as discussed.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Grisshammer, R., and Tate, C. G. (1995) Overexpression of integral membrane proteins for structural studies. Q. Rev. Biophys. 28, 315–422.
Loll, P. J. (2003) Membrane protein structural biology: the high throughput challenge. Journal of Structural Biology 142, 144–153.
Tate, C. G., and Grisshammer, R. (1996) Heterologous expression of G-protein-coupled receptors. Trends Biotechnol. 14, 426–430.
Wiener, M. C. (2004) A pedestrian guide to membrane protein crystallization. Methods 34, 364–372.
Selinsky, B. S. (ed.) (2003) Membrane Protein Protocols: Expression, Purification, and Characterization, Humana Press, Totowa, NJ.
Miroux, B., and Walker, J. E. (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J. Mol. Biol. 260, 289–298.
Sarramegna, V., Talmont, F., Demange, P., and Milon, A. (2003) Heterologous expression of G-protein-coupled receptors: comparison of expression systems from the standpoint of large-scale production and purification. Cell. Mol. Life Sci. 60, 1529–1546.
Tate, C. G. (2001) Overexpression of mammalian integral membrane proteins for structural studies. FEBS Lett. 504, 94–98.
Lemieux, M. J., Song, J., Kim, M. J., Huang, Y., Villa, A., Auer, M., et al. (2003) Three-dimensional crystallization of the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily. Protein Sci. 12, 2748– 2756.
Keyes, M. H., Gray, D. N., Kreh, K. E., and Sanders, C. R. (2003) Solubilizing detergents for membrane proteins, in (Iwata, S., ed.), Methods and Results in Crystallization of Membrane Proteins, International University Line, La Jolla, CA.
Dobrovetsky, E., Lu, M. L., Andorn-Broza, R., Khutoreskaya, G., Bray, J. E., Savchenko, A., et al. (2005) High throughput production of prokaryotic membrane proteins. J. Struct. Funct. Genomics 6, 33–50.
Lunin, V., Dobrovetsky, E., Khutoreskaya, G., Rongguang, Z., Joachimiak, A., Doyle, D. A., et al. (2006) Crystal structure of the CorA Mg2+ transporter. Nature 440, 833–837.
Lemberg, M. K., Menendez, J., Misik, A., Garcia, M., Koth, C. M., and Freeman, M. (2005) Mechanism of intramembrane proteolysis investigated with purified rhomboid proteases. EMBO J. 24, 464–472.
Urban, S., Lee, J. R., and Freeman, M. (2001) Drosophila rhomboid-1 defines a family of putative intramembrane serine proteases. Cell 107, 173–182.
McQuibban, G. A., Saurya, S., and Freeman, M. (2003) Mitochondrial membrane remodelling regulated by a conserved rhomboid protease. Nature 423, 537–541.
Cipolat, S., Rudka, T., Hartmann, D., Costa, V., Serneels, L., Craessaerts, K., et al. (2006) Mitochondrial rhomboid PARL regulates cytochrome c release during apoptosis via OPA1-dependent cristae remodeling. Cell 126, 163–175.
Gallio, M., Sturgill, G., Rather, P., and Kylsten, P. (2002) A conserved mechanism for extracellular signaling in eukaryotes and prokaryotes. Proc. Natl. Acad. Sci. USA 99, 12208–12213.
Brossier, F., Jewett, T. J., Sibley, L. D., and Urban, S. (2005) A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. USA 102, 4146–4151.
Nollert, P., Navarro, J., and Landau, E. M. (2002) Crystallization of membrane proteins in cubo. Methods Enzymol. 343, 183–199.
Nollert, P. (2004) Lipidic cubic phases as matrices for membrane protein crystallization. Methods 34, 348–353.
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., et al. (2000) The Protein Data Bank. Nucleic Acids Res. 28, 235–242.
Raman, P., Cherezov, V., and Caffrey, M. (2006) The Membrane Protein Data Bank. Cell. Mol. Life Sci. 63, 36–51.
Savchenko, A., Yee, A., Khachatryan, A., Skarina, T., Evdokimova, E., Pavlova, M., et al. (2003) Strategies for structural proteomics of prokaryotes: quantifying the advantages of studying orthologous proteins and of using both NMR and x-ray crystallography approaches. Proteins 50, 392–399.
Locher, K. P., Lee, A. T., and Rees, D. C. (2002) The E. coli BtuCD structure: a framework for ABC transporter architecture and mechanism. Science 296, 1091– 1098.
Jiang, Y., Lee, A., Chen, J., Ruta, V., Cadene, M., Chait, B. T., et al. (2003) X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41.
Chang, G., Spencer, R. H., Lee, A. T., Barclay, M. T., and Rees, D. C. (1998) Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mech-anosensitive ion channel. Science 282, 2220–2226.
Wasserman, J. D., Urban, S., and Freeman, M. (2000) A family of rhomboid-like genes: Drosophila rhomboid-1 and roughoid/rhomboid-3 cooperate to activate EGF receptor signaling. Genes Dev. 14, 1651–1663.
Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Savchenko, A., Cort, J. R., et al. (2000) Structural proteomics of an archaeon. Nat. Struct. Biol. 7, 903–909.
Moreland, N., Ashton, R., Baker, H. M., Ivanovic, I., Patterson, S., Arcus, V. L., et al. (2005) A flexible and economical medium-throughput strategy for protein production and crystallization. Acta Crystallogr. D Biol. Crystallogr. 61, 1378–1385.
Mohanty, A. K., Simmons, C. R., and Wiener, M. C. (2003) Inhibition of tobacco etch virus protease activity by detergents. Protein Expr. Purif. 27, 109–114.
Tucker, J., and Grisshammer, R. (1996) Purification of a rat neurotensin receptor expressed in Escherichia coli. Biochem. J. 317, 891–899.
Eshaghi, S., Hedren, M., Nasser, M. I., Hammarberg, T., Thornell, A., and Nordlund, P. (2005) An efficient strategy for high throughput expression screening of recombinant integral membrane proteins. Protein Sci. 14, 676–683.
Wang, D. N., Safferling, M., Lemieux, M. J., Griffith, H., Chen, Y., and Li, X. D. (2003) Practical aspects of overexpressing bacterial secondary membrane transporters for structural studies. Biochim. Biophys. Acta 1610, 23–36.
Hopkins, T. R. (1991) Physical and chemical cell disruption for the recovery of intracellular proteins, in (R. Seetharam, R. and Sharma, S. K., eds.), Purification and Analysis of Recombinant Proteins, Marcel Dekker, New York.
Iwata, S. (ed.) (2003) Methods and Results in Crystallization of Membrane Proteins. International University Line, La Jolla, CA.
Acknowledgments
The authors gratefully acknowledge many stimulating discussions with fellow researchers at Vertex Pharmaceuticals, the Ontario Centre for Structural Proteomics and the Structural Genomics Consortium. The authors are also grateful to J. Moore, A. Edwards, and D. Doyle for much advice and to M. Lemberg, J. Menendez, A. Misik, and M. Garcia for their work on Rhomboids. The authors are especially indebted to M. Freeman for the generous gift of several Rhomboid clones. C. M. K. conducted experiments on Rhomboids while at the Ontario Centre for Structural Proteomics (University of Toronto). This work was funded by Genome Canada and the Ontario Genomics Institute.
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
Willis, M.S., Koth, C.M. (2008). Structural Proteomics of Membrane Proteins: a Survey of Published Techniques and Design of a Rational High Throughput Strategy. 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_18
Download citation
DOI: https://doi.org/10.1007/978-1-60327-058-8_18
Publisher Name: Humana Press
Print ISBN: 978-1-58829-809-6
Online ISBN: 978-1-60327-058-8
eBook Packages: Springer Protocols