Abstract
We present a novel strategy for the connection of phenotype and genotype in vitro that can be used for the selection of functional proteins. The strategy involves the generation of a stable complex among a ribosome, an messenger RNA and its translated protein, without removal of the termination codon, as a result of the action of the ricin A chain during translation. The technique requires no transfection, no chemical synthesis, no ligation, and no removal of the termination codon. Thus, our novel ribosome-inactivation display system should provide, without loss of the pool population, a reliable, simple, and robust selection system for the in vitro evolution of the properties of proteins in a predictable direction by a combination of randomization and appropriate selection strategies.
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
Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994) Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455.
Kasahara, N., Dozy, A. M., and Kan, Y. M. (1994) Tissue-specific targeting of retroviral vectors through ligand-receptor interactions. Science 266, 1373–1376.
Georgiou, G., Poetschke, H. L., Stathopoulos, C., and Francisco, J. A. (1993) Practical applications of engineering Gram-negative bacterial cell surfaces. Trends Biotechnol. 11, 6–10.
Kieke, M. C., Cho, B. K., Boder, E. T., Kranz, D. M., and Wittrup, K. D. (1997) Isolation of anti-T cell receptor scFv mutants by yeast surface display. Protein Eng. 10, 1303–1310.
Mattheakis, L. C., Bhatt, R. R., and Dower, W. J. (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026.
Hanes, J. and Plückthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942.
He, M. Y., Menges, M., Groves, M. A. T., et al. (1999) Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J. Immunol. Methods 231, 105–117.
Roberts, R. W. and Szostak, J. W. (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. USA 94, 12,297–12,302.
Nemoto, N., Miyamoto-Sato, E., Husimi, Y., and Yanagawa, H. (1997) In vitro virus: Bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett. 414, 405–408.
Endo, Y. and Tsurugi, K. (1987) RNA N-glycosidase activity of ricin a chain. Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128–8130.
Endo, Y., Mitsui, K., Motizuki, M., and Tsuruki, K. (1987) The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28S ribosomal RNA caused by the toxins. J. Biol. Chem. 262, 5908–5912.
Moazed, D., Robertson, J., and Noller, H. (1989) Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S rRNA. Nature 334, 362–364.
Kudlicki, W., Kitaoka, Y., Odom, O. W., Kramer, G., and Hardesty, B. (1995) Elongation and folding of nascent ricin chains as peptidyl-tRNA on ribosomes: the effect of amino acid deletions on these processes. J. Mol. Biol. 252, 203–212.
Munishkin, A. and Wool, I. G. (1997) The ribosome-in-pieces: binding of elongation factor EF-G to oligoribonucleotides that mimic the sarcin/ricin and thiostrepton domains of 23S ribosomal RNA. Proc. Natl. Acad. Sci. USA 94, 12,280–12,284.
Eiklid, K., Olsnes, S., and Pihl, A. (1980) Entry of lethal doses of abrin, ricin, and modeccin into the cytosol of HeLa cells. Exp. Cell Res. 126, 321–326.
Mössner, E., Koch, H., and Plückthun, A. (2001) Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs. J. Mol. Biol. 308, 115–122.
Liu, R., Barrick, J. E., Szostak, J. W., and Roberts, R. W. (2000) Optimized synthesis of RNA-protein fusions for in vitro protein selection. Methods Enzymol. 318, 268–293.
Malkin, L. I. and Rich, A. (1967) Partial resistance of nascent polypeptide chains to proteolytic digestion due to ribosomal shielding. J. Mol. Biol. 26, 329–346.
Smith, W. P., Tai, P. C., and Davis, B. D. (1978) Interaction of secreted nascent chains with surrounding membrane in bacillus subtilis. Proc. Natl. Acad. Sci. USA 75, 5922–5925.
Komar, A. A., Kommer, A., Krasheninnikov, I. A., and Spirin, A. S. (1997) Cotranslational folding of globin. J. Biol. Chem. 272, 10,646–10,651.
Fedorov, A. N. and Baldwin, T. O. (1997) Cotranslational protein folding. J. Biol. Chem. 272, 32,715–32,718.
Kudlicki, W., Chirgwin, J., Kramer, G., and Hardesty, B. (1995) Folding of an enzyme into an active confirmation while bound as a peptidyl-tRNA to the ribosome. Biochemistry 34, 14,284–14,287.
Makeyev, E. V., Kolb, V. A., and Spirin, A. S. (1996) Enzymatic activity of the ribosome-bound nascent polypeptide. FEBS Lett. 378, 166–170.
Schaffitzel, C., Hanes, J., Jermutus, L., and Plückthun, A. (1999) Ribosome display: an in vitro method for selection and evolution of antibodies from libraries. J. Immunol. Methods 231, 119–135.
Wilson, D. S., Keefe, A. D., and Szostak, J. W. (2001) The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl. Acad. Sci. USA 98, 3750–3755.
Doi, N. and Yanagawa, H. (1999) STABLE: protein-DNA fusion system for screening of combinatorial protein libraries in vitro. FEBS Lett. 457, 227–230.
Cheadle, C., Ivashchenko, Y., South, V., et al. (1994) Identification of a Src SH3 domain binding motif by screening a random phage display library. J. Biol. Chem. 269, 24,034–24,039.
Gram, H., Schmitz, R., Zuber, J. F., and Baumann, G. (1997) Identification of phosphopeptide ligands for the Src-homology 2 (SH2) domain of Grb2 by phage display. Eur. J. Biochem. 246, 633–637.
Frankel, A., Welsh, P., Richardson, J., and Robertus, J. D. (1990) Role of arginine 180 and glutamic acid 177 of ricin toxin a chain in enzymatic inactivation of ribosomes. Mol. Cell Biol. 10, 6257–6263.
Kim, Y., Mlsa, D., Monzingo, A. F., Ready, M. P., Frankel, A., and Robertus, J. D. (1992) Structure of a ricin mutant showing rescue of activity by a noncatalytic residue. Biochemistry 31, 3294–3296.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Hanes, J., Jermutus, L., and Plücktun, A. (2000) Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol. 328, 404–430.
Sawata, Y. S., Wada, A., and Taira, K. (2003) An advanced ribosome-display with strengthened association (ARiSA) for in vitro selection of a peptide aptamer with strong affinity. Manuscript in preparation.
Sawata, S. Y., and Taira, K. (2003) Modified peptide selection in vitro by introduction of a protein-RNA interaction. Protein Eng. 16, 1115–1124.
Sawata, S. Y., Suyama, E., and Taira, K. (2004) A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro. Protein Eng. Des. Sel. 17, 501–508.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Fujita, S., Zhou, JM., Taira, K. (2007). Ribosome-Inactivation Display System. In: Arndt, K.M., Müller, K.M. (eds) Protein Engineering Protocols. Methods in Molecular Biology™, vol 352. Humana Press. https://doi.org/10.1385/1-59745-187-8:221
Download citation
DOI: https://doi.org/10.1385/1-59745-187-8:221
Publisher Name: Humana Press
Print ISBN: 978-1-58829-072-4
Online ISBN: 978-1-59745-187-1
eBook Packages: Springer Protocols