Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Improved Bivalent Miniantibodies, with Identical Avidity as Whole Antibodies, Produced by High Cell Density Fermentation of Escherichia coli

Bio/Technology volume 11, pages 1271–1277 (1993)Cite this article

Abstract

The combination of single-chain Fv-fragments (scFv) with a C-terminal, flexible linking region followed by a designed or natural dimerization domain provides a versatile system for targeted association of functional fragments in the periplasmic space of Escherichia coli. For homodimerization in vivo, two scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif form “miniantibodies” with significantly higher avidity than in the case of leucine zipper containing constructs. The favorable design probably results in an antiparallel four-helix bundle and brings the homodimer to the same avidity as the whole IgA antibody, from which the binding site was taken. The molecular weight of the bivalent miniantibody is almost the same as that of a monovalent Fab fragment. We report here a high-cell density fermentation of E. coli producing these miniantibodies and a work-up procedure suitable for large scale production. Without any need of subsequent chemical coupling in vitro, approximately 200 mg/l of functional dimeric miniantibodies can be directly obtained from the E. coli culture.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

References

  1. Pack, P. and Plückthun, A. 1992. Miniantibodies: use of amphipathic helices to produce functional, flexibly linked dimeric Fv fragments with high avidity in Escherichia coli . Biochemistry 31: 1579–1584.

    Article  CAS  Google Scholar 

  2. Crothers, D.M. and Metzger, H. 1972. The influence of polyvalency on the binding properties of antibodies. Immunochemistry 9: 341–357.

    Article  CAS  Google Scholar 

  3. Karush, F. 1978. The affinity of antibody: range, variability, and the role of multivalence, pp. 85–116. In: Immunoglobulins. Litman, G. W. and Good, R. A. (Eds.). Plenum Publishing Corp., NY and London.

    Chapter  Google Scholar 

  4. Glockshuber, R., Malia, M., Pfitzinger, I. and Plückthun, A. 1990. A comparison of strategies to stabilize immunoglobulin Fv-fragments. Biochemistry 29: 1362–1367.

    Article  CAS  Google Scholar 

  5. Bird, R.E., Hardman, K.D., Jacobson, J.W., Johnson, S., Kaufman, B.M., Lee, S.-M., Lee, T., Pope, S.H., Riordan, G.S. and Whitlow, M. 1988. Single-chain antigen-binding proteins. Science 242: 423–426.

    Article  CAS  Google Scholar 

  6. Huston, J.S., Levinson, D., Mudgett-Hunter, M., Tai, M.-S., Novotny, J., Margolies, M.N., Ridge, R.J., Bruccoleri, R.E., Haber, E., Crea, R. and Oppermann, H. 1988. Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli . Proc. Natl. Acad. Sci. USA 85: 5879–5883.

    Article  CAS  Google Scholar 

  7. O'Shea, E.K., Klemm, J.D., Kim, P.S. and Alber, T. 1991. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science 254: 539–545.

    Article  CAS  Google Scholar 

  8. O'Shea, E.K., Rutkowski, R. and Kim, P.S. 1989. Evidence that the leucine zipper is a coiled coil. Science 243: 538–542.

    Article  CAS  Google Scholar 

  9. Eisenberg, D., Wilcox, W., Eshita, S.M., Pryciak, P.M., Ho, S.P. and DeGrado, W.F. 1986. The design, synthesis, and crystallization of an alpha-helical peptide. Proteins 1: 16–22.

    Article  CAS  Google Scholar 

  10. Ho, S.P. and DeGrado, W.F. 1987. Design of a 4-helix bundle protein: synthesis of peptides which self-associate into a helical protein. J. Am. Chem. Soc. 109: 6751–6758.

    Article  CAS  Google Scholar 

  11. Glockshuber, R., Schmidt, T. and Plückthun, A. 1992. The disulfide bonds in antibody variable domains: effects on stability, folding in vitro, and functional expression in Escherichia coli . Biochemistry 31: 1270–1279.

    Article  CAS  Google Scholar 

  12. Satow, Y., Cohen, G.H., Padlan, E.A. and Davies, D.R. 1986. Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7Å. J. Mol. Biol. 190: 593–604.

    Article  CAS  Google Scholar 

  13. Skerra, A. and Plückthun, A. 1988. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli . Science 240: 1038–1041.

    Article  CAS  Google Scholar 

  14. Plückthun, A. and Skerra, A. 1989. Expression of functional Fv and Fab fragments in Escherichia coli . Meth. Enzymol. 178: 497–515.

    Article  Google Scholar 

  15. Pack, P., Knappik, A., Krebber, C. and Plückthun, A. 1992. Mono- and bivalent antibody fragments produced in E. coli : binding properties and folding in vivo, pp. 10–13. In: ACS Conference Proceedings Series, Harnessing Biotechnology for the 21st Century. Ladisch, M. R. and Bose, A. (Eds.) ACS, Washington, DC.

    Google Scholar 

  16. Wülfing, C. and Plückthun, A. 1993. A versatile and highly repressible E. coli expression system based on invertible promoters. Gene. In press

    Google Scholar 

  17. Riesenberg, D. 1991. High-cell-density cultivation of Escherichia coli . Current Opinion in Biotechnol. 2: 380–384.

    Article  CAS  Google Scholar 

  18. Metzger, H., Chesebro, B., Hadler, N.M., Lee, J. and Otchin, N. 1971. Modification of immunoglobulin combining sites, pp. 253–267. In: Progress in immunology: Proceedings of the 1st Congress of Immunology. Amos, B. (Ed.). Academic Press, New York.

    Chapter  Google Scholar 

  19. Blondel, A. and Bedouelle, H. 1991. Engineering the quaternary structure of an exported protein with a leucine zipper. Prot. Engineering 4: 457–461.

    Article  CAS  Google Scholar 

  20. Hu, J.C., O'Shea, E.K., Kim, P.S. and Sauer, R.T. 1990. Sequence requirements for coiled coils: analysis with λ-repressor-GCN4 leucine zipper fusions. Science 250: 1400–1403.

    Article  CAS  Google Scholar 

  21. Kostelny, S.A., Cole, M.S. and Tso, Y.J. 1992. Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 148: 1547–1553.

    CAS  PubMed  Google Scholar 

  22. Thomas, G.D., Chappell, M.J., Dykes, P.W., Ramsden, D.B., Godfrey, K.R., Ellis, J.R.M. and Bradwell, A.R. 1989. Effect of dose, molecular size, affinity, and protein uptake of antibody or ligand: a biomathematical model. Cancer Res. 49: 3290–3296.

    CAS  PubMed  Google Scholar 

  23. Huston, J.S., McCartney, J.S., Tai, M.-S., Mottola-Hartshorn, C., Jin, D., Warren, F., Keck, P. and Opperman, H. 1993. Medical applications of single-chain antibodies. Intern. Rev. of Immunol. In press

    Google Scholar 

  24. Maurer, R., Meyer, B.J. and Ptashne, M. 1980. Gene regulation at the right operator (O R) of bacteriophage λ-O R3 and autogenous negative control by represser. J. Mol. Biol. 139: 147–161.

    Article  CAS  Google Scholar 

  25. Carter, P., Kelley, R.F., Rodrigues, M.L., Snedecor, B., Covarrubias, M., Velligan, M.D., Wong, W.L.T., Rowland, A.M., Kotts, C.E., Carver, M.E., Yang, M., Bourell, J.H., Shepard, H.M. and Henner, D. 1992. High level Escherichia coli expression and production of a bivalent humanized antibody fragment. Bio/Technology 10: 163–167.

    CAS  Google Scholar 

  26. Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

    Google Scholar 

  27. Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, C.P., Urdal, D.L. and Conlon, P.J. 1988. A short polypeptide marker sequence useful for recombinant protein identification and purification. Bio/Technology 6: 1204–1210.

    Article  CAS  Google Scholar 

  28. Riesenberg, D., Menzel, K., Schulz, V., Schumann, K., Veith, G., Zuber, G. and Knorre, W.A. 1990. High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1. Appl. Microbiol. Biotechnol. 34: 77–82.

    Article  CAS  Google Scholar 

  29. Löser, C. 1990. Stabilitätsverhalten eines rekombinanten ColEl-Plasmid-derivates mit Ampicillin-Resistenzmarker und Human-Interferon-α-1-Gen in Escherichia coli . PhD-Thesis. Technical University Dresden, Fed. Rep. of Germany.

  30. Bignon, C. and Martin, P.-M. 1992. Improved sequencing of “mini-prep” double-stranded templates using a multiwell microplate system. Biotechniques 13: 104–106.

    Google Scholar 

  31. Neidhardt, F.C. 1987. Chemical composition of Escherichia coli. 3–6. In: Escherichia and Salmonella typhimurium. Cellular and Molecular Biology. F. C. Neidhardt (Ed. in chief). ASM Washington, D.C.

    Google Scholar 

  32. Lowry, O.H., Rosebrough, N.J., Fair, A.L. and Randall, R.J. 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: 265–275.

    CAS  Google Scholar 

  33. Layne, E. 1957. Spectrophotometric and turbidimetric methods for measuring proteins. Meth. Enzymol. 3: 447–454.

    Article  Google Scholar 

  34. Gill, S.T. and von Hippel, P.H. 1989. Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182: 319–326.

    Article  CAS  Google Scholar 

  35. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  Google Scholar 

  36. Burton, D.R. 1985. Immunoglobulin G: functional sites. Mol. Immunol. 22: 161–182.

    Article  CAS  Google Scholar 

  37. Dangl, J.L., Wensel, T.G., Morrison, S.L., Stryer, L., Herzenberg, L.A. and Oi, V.T. 1988. Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies. EMBO J. 7: 1989–1994.

    Article  CAS  Google Scholar 

  38. Lupas, A., Van Dyke, M. and Stock, J. 1991. Predicting coiled coils from protein sequences. Science 252: 1162–1164.

    Article  CAS  Google Scholar 

  39. Freund, C., Ross, A., Guth, B., Plückthun, A. and Holak, T. 1993. Characterization of the linker peptide of the single-chain Fv fragment of an antibody by NMR spectroscopy. FEBS Lett. 320: 97–100.

    Article  CAS  Google Scholar 

  40. Banner, D.W., Kokkinidis, M. and Tsernoglou, D. 1987. Structure of the ColE1 Rop protein at 1.7Å resolution. J. Mol. Biol. 196: 657–675.

    Article  CAS  Google Scholar 

  41. Kraulis, P.J. 1991. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24: 946–950.

    Article  Google Scholar 

Download references

Author information

Author notes

  1. Peter Pack

    Present address: MorphoSys, Frankfurter Ring 193a, D-80807, München, Fed. Rep. Germany

  2. Andreas Plückthun: Corresponding author. Address after October 1993: Biochemisches Institut, Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

Authors and Affiliations

  1. Max-Planck-Institut für Biochemie, Protein Engineering Group, Am Klopferspitz, D-82152, Martinsried, Fed. Rep. Germany

    Andreas Plückthun

  2. Hans-Knöll-Institut für Naturstoff-Forschung, Beutenbergstr. 11, D-07745, Jena, Fed. Rep. Germany

    Marian Kujau, Volker Schroeckh, Uwe Knüpfer, Rolf Wenderoth & Dieter Riesenberg

Authors
  1. Peter Pack
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Marian Kujau
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Volker Schroeckh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Uwe Knüpfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rolf Wenderoth
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Dieter Riesenberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Andreas Plückthun
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Pack, P., Kujau, M., Schroeckh, V. et al. Improved Bivalent Miniantibodies, with Identical Avidity as Whole Antibodies, Produced by High Cell Density Fermentation of Escherichia coli. Nat Biotechnol 11, 1271–1277 (1993). https://doi.org/10.1038/nbt1193-1271

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1193-1271

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing