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E. coliGene Expression Protocols pp 79–97Cite as

Calmodulin-Binding Peptide as a Removable Affinity Tag for Protein Purification

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Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 205))

Abstract

Protein purification is an important tool for investigations on protein function, structure analysis, and biotechnological use. Therefore a number of different techniques have been developed for fast, reliable, and reproducible overexpression and purification of relevant proteins. Affinity systems have been employed frequently due to speed, yield, and reduction of chromatographic steps necessary in order to get a highly purified protein. Over the years, different tags and matrices have been introduced to the scientific community, each providing a combination of advantages and disadvantages in the light of the protein of interest.

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References

  1. Vaillancourt, P., Simcox, T. G., and Zheng, C. F. (1997) Recovery of polypep-tides cleaved from purified calmodulin-binding peptide fusion proteins. Biotechniques 22, 45–453.

    Google Scholar 

  2. Zheng, C. F., Simcox, T., Xu, L., and Vaillancourt, P. (1997) A new expression vector for high level protein production, one step purification, and direct isotopic labeling of calmodulin-binding peptide fusion proteins. Gene 186, 55–60.

    Article  PubMed  CAS  Google Scholar 

  3. Vaillancourt, P., Zheng, C. F., Hoang, D. Q., and Breister, L. (2000) Affinity purification of recombinant proteins fused to calmodulin or to calmodulin-binding peptides. Methods Enzymol. 326, 340–362.

    Article  PubMed  CAS  Google Scholar 

  4. Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113–130.

    Article  PubMed  CAS  Google Scholar 

  5. Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D., et al. (1991) Interaction of the regulatory subunit (RII) of cAMP-dependent protein kinase with RII-anchoring proteins occurs through an amphipathic helix binding motif. J. Biol. Chem. 266, 14,188–14,192.

    PubMed  CAS  Google Scholar 

  6. Stofko-Hahn, R. E., Carr, D. W., and Scott, J. D. (1992) A single step purification for recombinant proteins. Characterization of a microtubule associated protein (MAP 2) fragment which associates with the type II cAMP-dependent protein kinase. FEBS Lett. 302, 274–278.

    Article  PubMed  CAS  Google Scholar 

  7. Maina, C. V., Riggs, P. D., Grandea, A. G., 3rd, et al. (1988) An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene 74, 365–373.

    Article  PubMed  CAS  Google Scholar 

  8. Klein, W., Winkelmann, D., Hahn, M., Weber, T., and Marahiel, M. A. (2000) Molecular characterization of the transition state regulator AbrB from Bacillus stearothermophilus. Biochim. Biophys. Acta 1493, 82–90.

    PubMed  CAS  Google Scholar 

  9. Aslanidis, C. and de Jong, P. J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069–6074.

    Article  PubMed  CAS  Google Scholar 

  10. Blanar, M. A. and Rutter, W. J. (1992) Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science 256, 1014–1018.

    Article  PubMed  CAS  Google Scholar 

  11. Phillips, T. A., VanBogelen, R. A., and Neidhardt, F. C. (1984) The lon gene product of Escherichia coli is a heat-shock protein. J. Bacteriol. 159, 283–287.

    PubMed  CAS  Google Scholar 

  12. 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.

    Article  PubMed  CAS  Google Scholar 

  13. Dubendorff, J. W. and Studier, F. W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J. Mol. Biol. 219, 45–59.

    Article  PubMed  CAS  Google Scholar 

  14. Dubendorff, J. W. and Studier, F. W. (1991) Creation of a T7 autogene. Cloning and expression of the gene for bacteriophage T7 RNA polymerase under control of its cognate promoter. J. Mol. Biol. 219, 61–68.

    Article  PubMed  CAS  Google Scholar 

  15. Wyborski, D. L., Bauer, J. C., Zheng, C. F., Felts, K., and Vaillancourt, P. (1999) An Escherichia coli expression vector that allows recovery of proteins with native N-termini from purified calmodulin-binding peptide fusions. Protein Expr. Purif. 16, 1–10.

    Article  PubMed  CAS  Google Scholar 

  16. Miller, J. H. (1992) A short course in bacterial genetics. A laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Labortory, Cold Spring Harbor, New York.

    Google Scholar 

  17. Chung, C. T., Niemela, S. L., and Miller, R. H. (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86, 2172–2175.

    Article  PubMed  CAS  Google Scholar 

  18. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1992) Short protocols in molecular biology. John Wiley & Sons, Harvard Medical School.

    Google Scholar 

  19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

    Google Scholar 

  20. Lämmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

    Article  Google Scholar 

  21. Bennett, J. and Scott, K. J. (1971) Quantitative staining of fraction I protein in polyacrylamide gels using Coomassie brillant blue. Anal. Biochem. 43, 173–182.

    Article  PubMed  CAS  Google Scholar 

  22. Bradford, M. M. (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.

    Article  PubMed  CAS  Google Scholar 

  23. Schägger, H. and Jagow, G. V. (1987) Tricine-sodium dodecyl sulfate-polyacry-lamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368–379.

    Article  PubMed  Google Scholar 

  24. Bloom, H., Beier, H., and Gross, H. S. (1987) Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels. Electrophoresis 8, 93–99.

    Article  Google Scholar 

  25. Wingfield, P. T. (1995) Preparation of soluble proteins from Escherichia coli. in Current protocols inprotein science, Vol. 1 (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds.), Wiley and Sons, New York.

    Google Scholar 

  26. Chong, Y. and Chen, H. (2001) Preparation of functional recombinant protein using a nondetergent sulfobetaine. BioTechniques 45, 24–26.

    Google Scholar 

  27. Wingfield, P. T., Palmer, I., and Liang, S.-M. (1995) Folding and purification of insoluble (inclusion-body) proteins from Escherichia coli. in Current protocols inprotein science, Vol. 1 (Coligan, J. E., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds.), Wiley and Sons, New York.

    Google Scholar 

  28. Neu, H. C. and Heppel, L. A. (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J. Biol. Chem. 240, 3685–3692.

    PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Institute for Pharmaceutical Biology, Rheinische Friedrich-Wilhelm University Bonn, Bonn, Germany

    Wolfgang Klein

Authors
  1. Wolfgang Klein
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Editor information

Editors and Affiliations

  1. Applied Molecular Evolution, San Diego, CA, USA

    Peter E. Vaillancourt

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© 2003 Humana Press Inc.

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Klein, W. (2003). Calmodulin-Binding Peptide as a Removable Affinity Tag for Protein Purification. In: Vaillancourt, P.E. (eds) E. coliGene Expression Protocols. Methods in Molecular Biology™, vol 205. Humana Press. https://doi.org/10.1385/1-59259-301-1:79

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  • DOI: https://doi.org/10.1385/1-59259-301-1:79

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-008-3

  • Online ISBN: 978-1-59259-301-9

  • eBook Packages: Springer Protocols

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