Transient Expression of Proteins Using COS Cells

Alejandro Aruffo1

1 Bristol‐Myers Squibb, Princeton, New Jersey
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.12
DOI:  10.1002/0471142727.mb1612s60
Online Posting Date:  November, 2002
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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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Table of Contents

  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Appropriate vector (see )
  • COS‐7 cells to be transfected (see ), adapted to growth in 2% serum (see annotation to step )
  • Dulbecco's 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 22)
  • DEAE‐dextran/chloroquine solution: PBS containing 10 mg/ml DEAE‐dextran (Sigma) + 2.5 mM chloroquine (Sigma)
  • 10% dimethyl sulfoxide (DMSO; Sigma) in PBS (see appendix 22 for 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 (unit 3.16), preparation of miniprep DNA (unit 1.6), purification of DNA by CsCl/ethidium bromide equilibrium centrifugation (unit 1.7), 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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Figures

Videos

Literature Cited

Literature Cited
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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 epression system for the production of proteins.
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