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Converting Monoclonal Antibodies into Fab Fragments for Transient Expression in Mammalian Cells

  • Joanne E. Nettleship
  • Aleksandra Flanagan
  • Nahid Rahman-Huq
  • Rebecca Hamer
  • Raymond J. OwensEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

In this chapter, protocols are described for converting mouse monoclonal antibodies into recombinant Fabs for transient expression in mammalian cells. Variable region genes are cloned by reverse transcription: PCR using either sequence specific or mixed 5′ primers that hybridise to the first framework sequence of the mouse light and heavy chains and 3′ primers that bind to the heavy- and light-chain constant regions. The amplified sequences are inserted into mammalian cell expression vectors by In-Fusion™ cloning. This method allows vector and amplified DNA sequences to be seamlessly joined in a ligation-independent reaction. Transient co-expression of light-chain and heavy-chain genes in HEK 293T cells enables production of recombinant Fabs for functional and structural studies.

Key words

HEK293T PCR cloning Fabs 

Notes

Acknowledgements

The Oxford Protein Production Facility is supported by grants from the Medical Research Council, UK, the Biotechnology and Biological Sciences Research Council, UK. AF is the recipient of a Wellcome Trust Research Studentship.

References

  1. 1.
    Kovari, L.C., Momany, C. and Rossmann, M.G. (1995) The use of antibody fragments for crystallization and structure determinations. Structure, 3, 1291–1293.Google Scholar
  2. 2.
    Hunte, C. and Münke, C. (2005) In Dingermann, T., Steinhilber, D. and Folkers, G. (eds.), Molecular biology in medicinal chemistry, pp. 300–322.Google Scholar
  3. 3.
    Buchegger, F., Haskell, C.M., Schreyer, M., Scazziga, B.R., Randin, S., Carrel, S. and Mach, J.P. (1983) Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice. J Exp Med, 158, 413–427.Google Scholar
  4. 4.
    King, D.J., Turner, A., Farnsworth, A.P., Adair, J.R., Owens, R.J., Pedley, R.B., Baldock, D., Proudfoot, K.A., Lawson, A.D., Beeley, N.R. et al. (1994) Improved tumor targeting with chemically cross-linked recombinant antibody fragments. Cancer Res, 54, 6176–6185.Google Scholar
  5. 5.
    Essono, S., Frobert, Y., Grassi, J., Creminon, C. and Boquet, D. (2003) A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA. J Immunol Methods, 279, 251–266.Google Scholar
  6. 6.
    Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H.R. and Pluckthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J Immunol Methods, 201, 35–55.Google Scholar
  7. 7.
    Wang, Z., Raifu, M., Howard, M., Smith, L., Hansen, D., Goldsby, R. and Ratner, D. (2000) Universal PCR amplification of mouse immunoglobulin gene variable regions: the design of degenerate primers and an assessment of the effect of DNA polymerase 3′ to 5′ exonuclease activity. J Immunol Methods, 233, 167–177.Google Scholar
  8. 8.
    Chardes, T., Villard, S., Ferrieres, G., Piechaczyk, M., Cerutti, M., Devauchelle, G. and Pau, B. (1999) Efficient amplification and direct sequencing of mouse variable regions from any immunoglobulin gene family. FEBS Lett, 452, 386–394.Google Scholar
  9. 9.
    Kettleborough, C.A., Saldanha, J., Ansell, K.H. and Bendig, M.M. (1993) Optimization of primers for cloning libraries of mouse immunoglobulin genes using the polymerase chain reaction. Eur J Immunol, 23, 206–211.Google Scholar
  10. 10.
    Ewert, S., Huber, T., Honegger, A. and Pluckthun, A. (2003) Biophysical properties of human antibody variable domains. J Mol Biol, 325, 531–553.Google Scholar
  11. 11.
    Plückthun, A., Krebber, A., Horn, U., Knüpfer, U., Wenderoth, R., Nieba, L., Proba, K. and Riesenberg, D. (1996) In McCafferty, J., Hoogenboom, H. R. and Chiswell, D. J. (eds.), Antibody engineering, a practical approach. Oxford University Press, Oxford, pp. 203–252.Google Scholar
  12. 12.
    Persson, M.A.A., Samuelsson, A., Yari, F. and Hinkula, J. (1996) Comparisons of expression in procaryotic and eucaryotic hosts of human recombinant Fab molecules. Immunotechnology, 2, 289.Google Scholar
  13. 13.
    Samuelsson, A., Yari, F., Hinkula, J., Ersoy, O., Norrby, E. and Persson, M.A.A. (1996) Human antibodies from phage libraries: neutralizing activity against human immunodeficiency virus type 1 equally improved after expression as Fab and IgG in mammalian cells. Eur J Immunol, 26, 3029–3034.Google Scholar
  14. 14.
    Sambrook, J. and Russell, D.W. (2001), Molecular cloning: a laboratory manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York, Vol. 3.Google Scholar
  15. 15.
    Iba, Y., Kaneko, T., Ekida, T., Miyata, K., Inouye, K., Kurosawa, Y. and Yasukawa, K. (1995) A new system for the expression of recombinant antibody in mammalian cells. Biotechnol Lett, 17, 135–138.Google Scholar
  16. 16.
    Nettleship, J.E., Ren, J., Rahman, N., Berrow, N.S., Hatherley, D., Barclay, A.N. and Owens, R.J. (2008) A pipeline for the production of antibody fragments for structural studies using transient expression in HEK 293T cells. Protein Expr Purif, 62, 83–89.Google Scholar
  17. 17.
    Frohman, M.A. (1993) Rapid amplification of complementary DNA ends for generation of full-length complementary DNAs: thermal RACE. Methods Enzymol, 218, 340–356.Google Scholar
  18. 18.
    Aricescu, A.R., Lu, W. and Jones, E.Y. (2006) A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallographica, Section D, 62, 1243–1250.Google Scholar
  19. 19.
    Burrows, P., LeJeune, M. and Kearney, J.F. (1979) Evidence that murine pre-B cells synthesise μ heavy chains but no light chains. Nature, 280, 838–840.Google Scholar
  20. 20.
    Morrison, S.L. and Scharff, M.D. (1975) Heavy chain-producing variants of a mouse myeloma cell line. J Immunol, 114, 655–659.Google Scholar
  21. 21.
    Nettleship, J.E., Rahman-Huq, N. and Owens, R.J. (2009) The production of glycoproteins by transient expression in Mammalian cells. Methods Mol Biol, 498, 245–263.Google Scholar
  22. 22.
    Strohal, R., Kroemer, G., Wick, G. and Kofler, R. (1987) Complete variable region sequence of a nonfunctionally rearranged kappa light chain transcribed in the nonsecretor P3-X63-Ag8.653 myeloma cell line. Nucleic Acids Res, 15, 2771.Google Scholar
  23. 23.
    Carroll, W.L., Mendel, E. and Levy, S. (1988) Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol Immunol, 25, 991–995.Google Scholar
  24. 24.
    Cochet, O., Martin, E., Fridman, W.H. and Teillaud, J.L. (1999) Selective PCR amplification of functional immunoglobulin light chain from hybridoma containing the aberrant MOPC 21-derived V kappa by PNA-mediated PCR clamping. Biotechniques, 26, 818–820, 822.Google Scholar
  25. 25.
    Carroll, W.L., Mendel, E. and Levy, S. (1988) Hybridoma fusion cell lines contain an aberrant kappa transcript. Mol Immunol, 25, 991–995.Google Scholar
  26. 26.
    Irani, Y., Tea, M., Tilton, R.G., Coster, D.J., Williams, K.A. and Brereton, H.M. (2008) PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts. J Immunol Methods, 336, 246–250.Google Scholar
  27. 27.
    Kutemeier, G., Harloff, C. and Mocikat, R. (1992) Rapid isolation of immunoglobulin variable genes from cell lysates of rat hybridomas by polymerase chain reaction. Hybridoma, 11, 23–32.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Joanne E. Nettleship
    • 1
  • Aleksandra Flanagan
    • 2
  • Nahid Rahman-Huq
    • 3
  • Rebecca Hamer
    • 4
  • Raymond J. Owens
    • 3
    Email author
  1. 1.Oxford Protein Production Facility UKResearch Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  2. 2.Division of Structural BiologyWellcome Trust Centre for Human GeneticsOxfordUK
  3. 3.Oxford Protein Production Facility UKResearch Complex at Harwell, Rutherford Appleton LaboratoryOxfordshireUK
  4. 4.Department of StatisticsUniversity of OxfordOxfordUK

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