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Optimizing the Production of Recombinant Prion Protein from CHO Cells

  • Sharon P. Bocking
  • Sarah E. Steane
  • Sureeta Saini
  • Alan D. Bennett
Chapter

Abstract

Recombinant aglycosyl murine prion protein (recPrP) has been over-expressed in a CHO cell line using the glutamine synthetase (GS) expression system. The recPrP lacks the glycophosphoinositol (GPI) anchor by which it is normally attached to the cell surface, and thus, the product is secreted into the medium. The aim of this work is to provide large quantities of mature length recPrP for structural and physicochemical studies. Currently, recPrP production is carried out in 500 mL spinner flask cultures, in which the cells are attached to Cytodex™ microcarriers. As serum interferes with the detection and purification of recPrP, our present production process includes a cell growth phase in medium containing 10% (v/v) dialysed foetal calf serum (dFCS), followed by a production phase in serum-free medium. These cultures yield up to 3 mg L-1 recPrP prior to purification. The concentration of mature length recPrP (23 kDa) usually increases over the first three days of the serum-free production phase, but then declines shortly after peak titres are attained. This loss of product in CHO cell cultures has been associated with the specific cleavage of the mature length prion protein into two fragments of 14 kDa and 9 kDa, not seen with bacterial (E. coli) host expression systems. Site specific cleavage not only compromises the productivity of the CHO cell system, but presents an obstacle in the preparation of homogeneous, mature length protein for x-ray crystallography studies. To date, attempts to control this problem with protease inhibitors have met with limited success. In this paper we provide evidence that site-specific cleavage of recPrP is a cell-associated event and describe a semi-continuous harvesting and medium replenishment approach to prevent accumulation of cleavage products in production cultures.

Keywords

Prion mammalian cells CHO cells site specific cleavage factorial design 

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Copyright information

© Springer Science+Business Media Dordrecht 2001

Authors and Affiliations

  • Sharon P. Bocking
    • 1
    • 2
  • Sarah E. Steane
    • 1
    • 2
  • Sureeta Saini
    • 1
    • 2
  • Alan D. Bennett
    • 1
    • 2
  1. 1.National Institute for Medical ResearchLondonUK
  2. 2.Institute for Animal HealthBerkshireUK

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