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Transient Expression of Proteins Using COS Cells

Alejandro Aruffo¹

¹Bristol‐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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Abstract

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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Basic Protocol
Commentary
Literature Cited
Figures

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Materials

Basic Protocol

Materials

Appropriate vector (see Background Information)
COS-7 cells to be transfected (see Critical Parameters), 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% CO₂ incubator
Phase-contrast microscope
Sorvall RT-6000B rotor (or equivalent)
Additional reagents and equipment for subcloning of DNA (cpmb unit 3.16), preparation of miniprep DNA (cpmb unit 1.6), purification of DNA by CsCl/ethidium bromide equilibrium centrifugation (cpmb unit 1.7), and flow cytometric analysis (Otten et al., 1995)

NOTE: All cell culture incubations should be carried out in a humidified 37°C, 6% CO₂ incubator unless otherwise stated.

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Figures

Figure 4.7.1

CDM8. CDM8 (Seed, 1987) is a COS cell expression/shuttle vector that contains an SV40-derived origin of replication (SV40 ori), eukaryotic transcription regulatory elements [splice and poly(A)⁺], a prokaryotic origin of replication (vx; derived from the pBR322 ori), and a prokaryotic genetic marker (supF). In addition, CDM8 contains an M13 origin of replication (M13 ori), a T7 RNA polymerase promoter (p_CMV/T7), and a polyoma-derived origin of replication (Py ori). Any restriction endonuclease sites shown can be used for cloning, but the inserted fragment must have its 5¢ end nearest the enchancer p_CMV/T7. The stuffer sequence is used to detect a size difference after restriction digestion.

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Literature Cited

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.
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	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.
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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.