Summary
Recently developed affinity purification methods have revolutionized our understanding of the higher-ordered structures of multisubunit, often low-abundance macromolecular complexes, including ribonucleoproteins (RNPs). Often, purification by classical, non-affinity-based techniques subjects salt-labile complexes to an ionic strength incompatible with the integrity of the RNP, leading to a misrepresentation of the true higher-ordered structure of these complexes. A family of plasmids has been generated that can be used to introduce a number of different epitope tags, including peptide-elutable affinity tags, into the genome of the yeast Saccharomyces cerevi-siae. Alternatively, these plasmids may be used for plasmid-borne expression of epitope-tagged proteins in either yeast or Escherichia coli. The gentle elution of the complex from the antibody affinity matrix can be performed at 4 °C and is compatible with a range of salt and pH conditions. RNPs purified by this method are active and suitable for downstream analyses such as RNA sequencing, structural analysis, or mass spectrometry peptide identification.
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References
Puig, O., Caspary, F., Rigaut, G., et al. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229.
Stevens, S. W. (2000) Analysis of low-abundance RNPs from yeast by affinity chromatography and mass spectrometry microsequencing. Methods Enzymol. 318, 385–398.
Wach, A., Brachat, A., AlbertiSegui, C., Rebischung, C., and Philippsen, P. (1997) Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast 13, 1065–1075.
Barnes, W. M. (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. U. S. A. 91, 2216–2220.
Gietz, R. D., and Woods, R. A. (2002) Transformation of yeast by lithium acetate/ single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350, 87–96.
Baker-Brachmann, C., Davies, A., Cost, G. J., et al. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132.
Sherman, F. (1991) Getting started with yeast. Methods Enzymol. 194, 3–21.
Yaffe, M. P., and Schatz, G. (1984) Two nuclear mutations that block mitochon-drial import in yeast. Proc. Natl. Acad. Sci. U. S. A. 81, 4819–4823.
Grussenmeyer, T., Scheidtmann, K. H., Hutchinson, M. A., Eckhart W., and Walter, G. (1985) Complexes of polyoma-virus medium T-antigen and cellular proteins. Proc. Natl. Acad. Sci. U. S. A. 82, 7952–7954.
Ganderton, R. H., Stanley, K. K., Field, C. E., et al. (1992) A monoclonal antipeptide antibody reacting with the insulin-receptor β-subunit. Biochem. J. 288, 195–205.
Bradford, M. (1976) A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein dye-binding. Anal. Biochem. 72, 248–254.
Ansari, A., and Schwer, B. (1995) SLU7 and a novel activity, SSF1, act during the PRP16-dependent step of yeast pre-mRNA splicing. EMBO J. 14, 4001–4009.
Stevens, S. W., and Abelson, J. (2002) Yeast pre-mRNA splicing: Methods, mechanisms, and machinery. Methods Enzymol. 351, 200ȓ220.
Bouveret, E., Rigaut, G., Shevchenko, A., Wilm, M., and Séraphin, B. (2000) A Sm-like protein complex that participates in mRNA degradation. EMBO J. 19, 1661–1671.
Bach, M., Winkelmann, G., and Lührmann, R. (1989) 20S small nuclear ribonu-cleoprotein U5 shows a surprisingly complex protein composition. Proc. Natl. Acad. Sci. U. S. A. 86, 6038–6042.
Behrens, S. E., and Lührmann, R. (1991) Immunoaffinity purification of a [U4/U6●U5] tri-snRNP from human-cells. Genes Dev. 5, 1439–1452.
Stevens, S. W., Barta, I., Ge, H. Y., et al. (2001) Biochemical and genetic analyses of the U5, U6 and U4/U6●U5 small nuclear ribonucleoproteins from Saccharomyces cerevisiae. RNA.7, 1543–1553.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY.
Neuhoff, V., Arold, N., Taube, D., and Ehrhardt, W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262.
Blum, H., Hildburg, B., and Gross, H. J. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis.8, 93–99.
Davis, M. T., and Lee, T. D. (1997) Variable flow liquid chromatography tandem mass spectrometry and the comprehensive analysis of complex protein digest mixtures. J. Am. Soc. Mass Spectrom. 8, 1059–1069.
Davis, M. T., and Lee, T. D. (1998) Rapid protein identification using a microscale electrospray LC/MS system on an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 9, 194–201.
Eng, J. K., McCormack, A. L., and Yates, J. R. (1994) An approach to correlate tandem mass-spectral data of peptides with amino-acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 876–989.
Link, A. J., Eng, J., Schieltz, D. M., et al. (1999) Direct analysis of protein complexes using mass spectrometry. Nature Biotechnol. 17, 676–682.
Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 185, 60–89.
Acknowledgments
I gratefully acknowledge the support of John Abelson, in whose laboratory these techniques were developed. I thank Christine Guthrie and Amy Kistler for suggesting the polyoma epitope and for the contribution of the hybridoma cells. This work is supported by a grant from the Welch Foundation (F-1564), the National Science Foundation (MCB-0448556), and the American Cancer Society (RSG-05-137-01-MCB).
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Stevens, S.W. (2008). Purification of Ribonucleoproteins Using Peptide-Elutable Antibodies and Other Affinity Techniques. In: Lin, RJ. (eds) RNA-Protein Interaction Protocols. Methods in Molecular Biology, vol 488. Humana Press. https://doi.org/10.1007/978-1-60327-475-3_5
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DOI: https://doi.org/10.1007/978-1-60327-475-3_5
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