Transient Expression of Proteins Using COS Cells

Alejandro Aruffo1

1 Bristol‐Myers Squibb, Princeton, New Jersey
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 4.7
DOI:  10.1002/0471142301.ns0407s02
Online Posting Date:  May, 2001
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This unit describes the use of COS cells to efficiently produce a desired protein in a short period of time. These cells express high levels of the SV40 large tumor (T) antigen, which is necessary to initiate viral DNA replication at the SV40 origin. Each COS cell transfected with DNA encoding a cell‐surface antigen (in the appropriate vector) or cytoplasmic protein will express several thousand to several hundred thousand copies of the protein 72 hr posttransfection. If the transfected DNA encodes a secreted protein, up to 10 mg of protein can be recovered from the supernatant of the transfected COS cells 1 week posttransfection. COS cell transient expression systems have also been used to screen cDNA libraries, to isolate cDNAs encoding cell‐surface proteins, secreted proteins, and DNA binding proteins, and to test protein expression vectors rapidly prior to the preparation of stable cell lines.

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • Appropriate vector (see )
  • COS‐7 cells to be transfected (see ), adapted to growth in 2% serum (see annotation to step )
  • Dulbeccos minimum essential medium (DMEM) with 2% calf serum (DMEM‐2 CS)
  • DMEM with 2% NuSerum (Collaborative Research; DMEM‐2 NS), 37°C
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • DEAE‐dextran/chloroquine solution:PBS containing 10 mg/ml DEAE‐dextran (Sigma) + 2.5 mM chloroquine (Sigma)
  • 10% dimethyl sulfoxide (DMSO; Sigma) in PBS
  • 0.5 mM EDTA in PBS
  • 100‐mm tissue culture dishes
  • Humidified 37°C, 6% CO 2 incubator
  • Phase‐contrast microscope
  • Sorvall RT‐6000B rotor (or equivalent)
  • Additional reagents and equipment for subcloning of DNA (CPMB UNIT ), preparation of miniprep DNA (CPMB UNIT ), purification of DNA by CsCl/ethidium bromide equilibrium centrifugation (CPMB UNIT ), and flow cytometric analysis (Otten et al., )
NOTE: All cell culture incubations should be carried out in a humidified 37°C, 6% CO 2 incubator unless otherwise stated.
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Literature Cited

   Aruffo, A. and Seed, B. 1987a. Molecular cloning of a CD28 cDNA by a high‐efficiency COS cell expression system. Proc. Natl. Acad. Sci. U.S.A. 84:8573‐8577.
   Aruffo, A. and Seed, B. 1987b. Molecular cloning of two CD7 (T cell leukemia antigen) cDNAs by a COS cell expression system. EMBO J. 6:3313‐3316.
   Aruffo, A., Stamenkovic, I., Melnick, M., Underhill, C.B., and Seed, B. 1990. CD44 is the principal cell‐surface receptor for hyaluronate. Cell 61:1303‐1313.
   Dailey, L. and Basilico, C.J. 1985. Sequences in the polyoma virus DNA regulatory region involved in viral DNA replication and early gene expression. J. Virol. 54:739‐749.
   Gluzman, Y. 1981. SV‐40 transformed simian cells support the replication of early SV40 mutants. Cell 23:175‐182.
   Goelz, S.E., Hession, C., Goff, D., Griffiths, B., Tizard, R., Newman, B., Chi‐Rosso, G., and Lobb, R. 1990. ELFT: A gene that directs the expression of an ELAM‐1 ligand. Cell 63:1349‐1356.
   Kaufman, R.J. 1985. Identification of the components necessary for adenovirus translational control and their utilization in cDNA expression vectors. Proc. Natl. Acad. Sci. U.S.A. 82:689‐693.
   Laub, O. and Rutter, W.J. 1983. Expression of the human insulin gene and cDNA in a heterologous mammalian system. J. Biol. Chem. 258:6043‐6050.
   Lusky, M. and Botchan, M. 1981. Inhibition of SV40 replication in simian cells by specific pBR322 DNA sequences. Nature 293:79‐81.
   Mellon, P., Parker, V., Gluzman, Y., and Maniatis, T. 1981. Identification of DNA sequences required for transcription of the human α1‐globin gene in a new SV40 host‐vector system. Cell 27:279‐288.
   Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto, Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom, J., Takahashi, T., Kuno, M., and Numa, S. 1984. Expression of functional acetylcholine receptor from cloned cDNAs. Nature 307:604‐608.
   Otten, G., Yokoyama, W.M. and Holmes, K.H. 1995. Flow cytometry analysis using the Becton Dickinson FACScan. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach, and W. Strober, eds.) pp. 5.4.1‐5.4.19. John Wiley & Sons, New York.
   Rose, J.K. and Bergmann, J.E. 1982. Expression from cloned cDNA of cell‐surface, secreted forms of the glycoprotein of vesicular stomatitis virus in eucaryotic cells. Cell 30:753‐ 62.
   Seed, B. 1987. An LFA‐3 cDNA encodes a phospholipid‐linked membrane protein homologous to its receptor CD2. Nature 329:840‐842.
   Seed, B. and Aruffo, A. 1987. Molecular cloning of the CD2 antigen, the T cell erythrocyte receptor, by a rapid immunoselection procedure. Proc. Natl. Acad. Sci. U.S.A. 84:3365‐3369.
   Sims, J.E., March, C.J., Cosman, D., Widmer, M.B., MacDonald, H.R., McMahan, C.J., Grubin, C.E., Wignall, J.M., Jackson, J.L., Call, S.M., Friend, D., Alpert, A.R., Gillis, S., Urdal, D.L., and Dower, S.K. 1988. cDNA expression cloning of the IL‐1 receptor, a member of the immunoglobulin superfamily. Science 241:585‐589.
   Tsai, F., Martin, D.I.K., Zon, L.I., D'Andrea, D., Wong, G.G., and Orkin, S.H. 1989. Cloning of cDNA for the major DNA‐binding protein of the erythroid lineage through expression in mammalian cells. Nature 339:446‐451.
   Warren, T.G. and Shields, D. 1984. Expression of preprosomatostatin in heterologous cells: Biosynthesis, posttranslational processing, and secretion of mature somatostatin. Cell 39:547‐555.
   Wong, G.G., Witek, J.S., Temple, P.A., Wilkens, K.M., Leary, A.C., Luxenber, D.P., Jones, S.S., Brown, E.L., Kay, R.M., Orr, E.C., Shoemaker, C., Golde, D.W., Kaufman, R.J., Hewick, R.M., Wang, E.A., and Clark, S.C. 1985. Human GM‐CSF: Molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science 228:810‐815.
   Yang, Y., Ciarletta, A.B., Temple, P.A., Chung, M.P., Kovacic, S., Witek‐Giannotti, J.S., Leary, A.C., Kriz, R., Donahue, R.E., Wong, G.G., and Clark, S.C. 1986. Human IL‐3 (multi‐CSF): Identification by expression cloning of a novel hematopoietic growth factor related to murine IL‐3. Cell 47:3‐10.
Key Reference
   Warren and Shields, 1984. See above.
  This article shows that COS cells can be used as an efficient, short‐term, mammalian expression system for the production of proteins.
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