Advertisement

Tools for Coproducing Multiple Proteins in Mammalian Cells

  • Zahra Assur
  • Wayne A. Hendrickson
  • Filippo ManciaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 801)

Abstract

Structural and functional studies of many mammalian systems are critically dependent on abundant supplies of recombinant multiprotein complexes. Mammalian cells are often the most ideal, if not the only suitable host for such experiments. This is due to their intrinsic capability to generate functional mammalian proteins. This advantage is frequently countered by problems with yields in expression, time required to generate overexpressing lines, and elevated costs. Coexpression of multiple proteins adds another level of complexity to these experiments, as cells need to be screened and selected for expression of suitable levels of each component. Here, we present an efficient fluorescence marking procedure for establishing stable cell lines that overexpress two proteins in coordination, and we validate the method in the production of recombinant monoclonal antibody Fab fragments. This procedure may readily be expanded to systems of greater complexity, comprising more than two components.

Key words

Coexpression Monoclonal antibody Fab fragments Protein complexes Protein–protein interactions Mammalian cells 

Notes

Acknowledgments

We are grateful to Richard Axel for discussions and to Ira Schieren for sharing with us his invaluable expertise on cell sorting. This work was supported in part by NIH grants GM68671 and GM75026.

References

  1. 1.
    Gavin, A. C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L. J., Bastuck, S., Dumpelfeld, B., Edelmann, A., Heurtier, M. A., Hoffman, V., Hoefert, C., Klein, K., Hudak, M., Michon, A. M., Schelder, M., Schirle, M., Remor, M., Rudi, T., Hooper, S., Bauer, A., Bouwmeester, T., Casari, G., Drewes, G., Neubauer, G., Rick, J. M., Kuster, B., Bork, P., Russell, R. B., and Superti-Furga, G. (2006) Proteome survey reveals modularity of the yeast cell machinery, Nature 440, 631–636.Google Scholar
  2. 2.
    Krogan, N. J., Cagney, G., Yu, H., Zhong, G., Guo, X., Ignatchenko, A., Li, J., Pu, S., Datta, N., Tikuisis, A. P., Punna, T., Peregrin-Alvarez, J. M., Shales, M., Zhang, X., Davey, M., Robinson, M. D., Paccanaro, A., Bray, J. E., Sheung, A., Beattie, B., Richards, D. P., Canadien, V., Lalev, A., Mena, F., Wong, P., Starostine, A., Canete, M. M., Vlasblom, J., Wu, S., Orsi, C., Collins, S. R., Chandran, S., Haw, R., Rilstone, J. J., Gandi, K., Thompson, N. J., Musso, G., St Onge, P., Ghanny, S., Lam, M. H., Butland, G., Altaf-Ul, A. M., Kanaya, S., Shilatifard, A., O’Shea, E., Weissman, J. S., Ingles, C. J., Hughes, T. R., Parkinson, J., Gerstein, M., Wodak, S. J., Emili, A., and Greenblatt, J. F. (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae, Nature 440, 637–643.Google Scholar
  3. 3.
    Romier, C., Ben Jelloul, M., Albeck, S., Buchwald, G., Busso, D., Celie, P. H., Christodoulou, E., De Marco, V., van Gerwen, S., Knipscheer, P., Lebbink, J. H., Notenboom, V., Poterszman, A., Rochel, N., Cohen, S. X., Unger, T., Sussman, J. L., Moras, D., Sixma, T. K., and Perrakis, A. (2006) Co-expression of protein complexes in prokaryotic and eukaryotic hosts: experimental procedures, database tracking and case studies, Acta Crystallogr D Biol Crystallogr 62, 1232–1242.Google Scholar
  4. 4.
    Tan, S. (2001) A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli, Protein Expr Purif 21, 224–234.Google Scholar
  5. 5.
    Yin, J., Li, G., Ren, X., and Herrler, G. (2007) Select what you need: a comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes, J Biotechnol 127, 335–347.Google Scholar
  6. 6.
    Geisse, S., Gram, H., Kleuser, B., and Kocher, H. P. (1996) Eukaryotic expression systems: a comparison, Protein Expr Purif 8, 271–282.Google Scholar
  7. 7.
    Eifler, N., Duckely, M., Sumanovski, L. T., Egan, T. M., Oksche, A., Konopka, J. B., Luthi, A., Engel, A., and Werten, P. J. (2007) Functional expression of mammalian receptors and membrane channels in different cells, J Struct Biol 159, 179–193.Google Scholar
  8. 8.
    Lundstrom, K. (2003) Semliki Forest virus vectors for rapid and high-level expression of integral membrane proteins, Biochim Biophys Acta 1610, 90–96.Google Scholar
  9. 9.
    Birch, J. R., and Racher, A. J. (2006) Antibody production, Adv Drug Deliv Rev 58, 671–685.Google Scholar
  10. 10.
    Assur, Z., Schieren, I., Hendrickson, W. A., and Mancia, F. (2007) Two-color selection for amplified co-production of proteins in mammalian cells, Protein Expr Purif 55, 319–324.Google Scholar
  11. 11.
    Vagner, S., Galy, B., and Pyronnet, S. (2001) Irresistible IRES. Attracting the translation machinery to internal ribosome entry sites, EMBO Rep 2, 893–898.Google Scholar
  12. 12.
    Tsien, R. Y. (1998) The green fluorescent protein, Annu Rev Biochem 67, 509–544.Google Scholar
  13. 13.
    Knop, M., Barr, F., Riedel, C. G., Heckel, T., and Reichel, C. (2002) Improved version of the red fluorescent protein (drFP583/DsRed/RFP), Biotechniques 33, 592–602.Google Scholar
  14. 14.
    Thomsen, D. R., Stenberg, R. M., Goins, W. F., and Stinski, M. F. (1984) Promoter-regulatory region of the major immediate early gene of human cytomegalovirus, Proc Natl Acad Sci U S A 81, 659–663.Google Scholar
  15. 15.
    Mancia, F., Patel, S. D., Rajala, M. W., Scherer, P. E., Nemes, A., Schieren, I., Hendrickson, W. A., and Shapiro, L. (2004) Optimization of protein production in mammalian cells with a coexpressed fluorescent marker, Structure (Camb) 12, 1355–1360.Google Scholar
  16. 16.
    Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments, Gene 141, 79–84.Google Scholar
  17. 17.
    Julius, D., MacDermott, A. B., Axel, R., and Jessell, T. M. (1988) Molecular characterization of a functional cDNA encoding the serotonin 1c receptor, Science 241, 558–564.Google Scholar
  18. 18.
    Mancia, F., Brenner-Morton, S., Siegel, R., Assur, Z., Sun, Y., Schieren, I., Mendelsohn, M., Axel, R., and Hendrickson, W. A. (2007) Production and characterization of monoclonal antibodies sensitive to conformation in the 5HT2c serotonin receptor, Proc Natl Acad Sci U S A 104, 4303–4308.Google Scholar
  19. 19.
    Amit, A. G., Mariuzza, R. A., Phillips, S. E., and Poljak, R. J. (1986) Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution, Science 233, 747–753.Google Scholar
  20. 20.
    Ali, S. H., and DeCaprio, J. A. (2001) Cellular transformation by SV40 large T antigen: interaction with host proteins, Semin Cancer Biol 11, 15–23.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Zahra Assur
    • 1
  • Wayne A. Hendrickson
    • 1
  • Filippo Mancia
    • 2
    Email author
  1. 1.Department of Biochemistry and Molecular BiophysicsColumbia UniversityNew YorkUSA
  2. 2.Department of Physiology and Cellular BiophysicsColumbia UniversityNew YorkUSA

Personalised recommendations