Abstract
Chemically synthesized truncated Ras proteins are coupled to C-terminal Ras peptides via a maleimidocaproyl linker. The resulting product is isolated by extraction with Triton X-114. The biological activity of these oncogenic Ras lipoproteins can be determined in a cell-based differentiation assay by microinjection into PC12 cells.
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
Takai, Y., Sasaki, T., and Matozaki, T. (2001) Small GTP-binding proteins. Physiol. Rev. 81, 153–208.
Roy, M. O., Leventis, R., and Silvius, J. R. (2000) Mutational and biochemical analysis of plasma membrane targeting mediated by the farnesylated, polybasic carboxy terminus of K-Ras4B. Biochemistry 39, 8298–8307.
Gelb, M. H. (1997) Protein biochemistry—protein prenylation, et cetera—signal transduction in two dimensions. Science 275, 1750–1751.
Bartels, D. J., Mitchell, D. A., Dong, X. W., and Deschenes, R. J. (1999) Erf2, a novel gene product that affects the localization and palmitoylation of Ras2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 19, 6775–6787.
Lobo, S., Greentree, W. K., Linder, M. E., and Deschenes, R. J. (2002) Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J Biol. Chem. 277, 41,268–41,273.
Kuhlmann, J. and Herrmann, C. (2001) Biophysical characterization of the Ras protein. Topics Curr. Chem. 211, 61–116.
Page, M. J., Hall, A., Rhodes, S., Skinner, R. H., Murphy, V., Sydenham, M., and Lowe, P. N. (1989) Expression and characterization of the Ha-Ras p21 protein produced at high levels in the insect/baculovirus system. J Biol. Chem. 264, 19,147–19,154.
Rubio, I., Wittig, U., Meyer, C., Heinze, R., Kadereit, D., Waldmann, H., Downward, L., and Wetzker, R. (1999) Farnesylation of Ras is important for the interaction with phosphoinositide 3-kinase gamma. Eur. J. Biochem. 266, 70–82.
Inouye, K., Mizutani, S., Koide, H., and Kaziro, Y. (2000) Formation of the Ras dimer is essential for Raf-1 activation. J. Biol. Chem. 275, 3737–3740.
Zimmerman, K. K., Scholten, J. D., Huang, C.-C., Fierke, C. A., and Hupe, D. J. (1998) High-level expression of rat farnesyl: Protein transferase in Escherichia coli as a translationally coupled heterodimer. Protein Expression Purification 14, 395–402.
Seabra, M. C. and James, G. L. (1998) Prenylation assays for small GTPases, in Transmembrane Signaling Protocols (Bar-Sagi, D., ed.), Humana Press, Totowa, NJ, pp. 251–260.
Bader, B., Kuhn, K., Owen, D. J., Waldmann, H., Wittinghofer, A., and Kuhlmann, J. (2000) Bioorganic synthesis of lipid-modified proteins for the study of signal transduction. Nature 403, 223–226.
Bordier, C. (1981) Phase separation of integral membrane proteins in Triton X-114 solution. J. Biol. Chem. 256, 1604–1607.
Bar-Sagi, D. and Feramisco, J. R. (1985) Microinjection of the Ras oncogene protein into PC12 cells induces morphological differentiation. Cell 42, 841–848.
Kuhn, K., Owen, D. J., Bader, B., Wittinghofer, A., Kuhlmann, J., and Waldmann, H. (2001) Synthesis of functional Ras lipoproteins and fluorescent derivatives. J. Am. Chem. Soc. 123, 1023–1035.
Kuhlmann, J., Tebbe, A., Volkert, M., Wagner, M., Uwai, K., and Waldmann, H., (2002) Photoactivatable synthetic Ras proteins: “Baits” for the identification of plasma-membrane-bound binding partners of Ras. Ang. Chem. Int. Ed. 41, 2546–2550.
Masterson, W. J. and Magee, A. I. (1992) Lipid modifications involved in protein targeting, in Protein Targeting: A Practical Approach (Magee, A. I. and Wileman, T., eds.), IRL Press at Oxford University Press, New York, NY, p. 242.
Greene, L. A. and Tischler, A. S. (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc. Natl. Acad. Sci. USA 73, 2424–2428.
Schmidt, G. and Wittinghofer, A. (2000) Priming of PC12 cells for semiquantitative microinjection studies involving Ras. FEBS Lett. 474, 184–188.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Wagner, M., Kuhlmann, J. (2004). Generation and Characterization of Ras Lipoproteins Based on Chemical Coupling. In: Niemeyer, C.M. (eds) Bioconjugation Protocols. Methods in Molecular Biology™, vol 283. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-813-7:245
Download citation
DOI: https://doi.org/10.1385/1-59259-813-7:245
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-58829-098-4
Online ISBN: 978-1-59259-813-7
eBook Packages: Springer Protocols