Purification and Analysis of Strep-Tagged Antibody-Fragments

Part of the Springer Lab Manuals book series (SLM)

Abstract

The development of generic purification techniques for immunoglobulin (Ig) fragments has gained considerable interest, particularly because the corresponding antigens are often too scarce or unstable to prepare a matrix for affinity chromatography. In this respect, the use of a short peptide tag with defined molecular recognition properties has the advantage that it should not interfere with the function of the antibody fragment and, therefore, its removal is not necessary for most in vitro applications. The Streptag constitutes a nine-amino acid peptide with the sequence “Ala-TrpArg-His-Pro-Gln-Phe-Gly-Gly“, which can easily be fused to scFv, Fv, and Fab fragments. This peptide confers reversible binding activity towards the well known protein-reagent streptavidin. Hence, it enables the purification of a corresponding fusion protein via streptavidin affinity chromatography in one step. Furthermore, the Strep-tag can be used for detection on Western blots or in ELISAs using streptavidin-enzyme conjugates.

Keywords

Crystallization Urea Electrophoresis Fractionation Peri 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Blake MS, Johnston KH, Russel-Jones GJ, Gotschlich EC (1984) A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots. Anal Biochem 136: 175–179PubMedCrossRefGoogle Scholar
  2. Boulot G, Eisele JL, Bentley GA, Bhat TN, Ward ES, Winter G and Poljak RJ (1990) Crystallization and preliminary X-ray diffraction study of the bacterially expressed Fv from the monoclonal anti-lysozyme antibody D1.3 and of its complex with the antigen, lysozyme. J Mol Biol 213: 617–619PubMedCrossRefGoogle Scholar
  3. Fling SP, Gregerson DS (1986) Peptide and protein molecular weight determination by electrophoresis using a high-molarity Tris-buffer system without urea. Anal Biochem 155: 83–88PubMedCrossRefGoogle Scholar
  4. Ostermeier C, Iwata S, Ludwig B and Michel H (1995) Fv fragment-mediated crystallization of the membrane protein bacterial cytochrome c oxidase. Nature Struct Biol 2: 842–846PubMedCrossRefGoogle Scholar
  5. Ostermeier C, Harrenga A, Ermler U and Michel H (1997) Structure at a 2.7 A resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody Fv fragment. Proc Natl Acad Sci USA 94: 10547–10553PubMedCrossRefGoogle Scholar
  6. Schiweck W, Buxbaum B, Schätzlein C, Neiss HG, Skerra A (1997) Sequence analysis and bacterial production of the anti-c-myc antibody 9E10: the VH domain has an extended CDR-H3 and exhibits unusual solubility. FEBS Lett 414: 33–38PubMedCrossRefGoogle Scholar
  7. Schmidt TGM and Skerra A (1993) The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Eng 6: 109–122PubMedCrossRefGoogle Scholar
  8. Schmidt TGM and Skerra A (1994) One-step affinity purification of bacterially produced proteins by means of the “Strep tag” and immobilized recombinant core streptavidin. J Chromatogr A 676: 337–345PubMedCrossRefGoogle Scholar
  9. Schmidt TGM, Koepke J, Frank R, and Skerra A (1996) Molecular interaction between the Strep-tag affinity peptide and ist cognate target, streptavidin. J Mol Biol 255: 753–766PubMedCrossRefGoogle Scholar
  10. Skerra A (1993) Bacterial expression of immunglobulin fragments. Curr Opin Immunol 5: 256–262PubMedCrossRefGoogle Scholar
  11. Skerra A (1994a) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141: 79–84PubMedCrossRefGoogle Scholar
  12. Skerra A (1994b) Use of the tetracycline promotor for the tightly regulated production of a murine antibody fragment in Escherichia coli. Gene 151: 131–135PubMedCrossRefGoogle Scholar
  13. Skerra A and Plückthun A (1988) Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science 240: 1038–1041PubMedCrossRefGoogle Scholar
  14. Voss S and Skerra A (1997) Mutagenesis of a flexible loop in streptavidin leads to higher affinity for the Strep-tag II peptide and improved performance in recombinant protein purification. Protein Eng 10: 975–982PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2001

Authors and Affiliations

  1. 1.Lehrstuhl für Biologische ChemieTechnische Universität MünchenFreising-WeihenstephanGermany

Personalised recommendations