Abstract
We report here the successful scale up of transient recombinant protein expression to litre scale using Semliki Forest Virus System. The expression of bacterial β-galactosidase was initially compared in BHK and CHO cells and the conditions for optimal infection of BHK cells were identified. 10% FCS in a medium at pH 6.9 and infection in small volumes were found to be optimal. A high MOI results in an increased recombinant protein yield. Stirring does not affect the infection process. Finally we applied these optimal conditions to the production of a microsomal enzyme, human cyclooxygenase-2 in suspension spinners. Five independant productions at the 1 litre scale yielded reproducible substantial amounts of recombinant protein (16 mg microsomal protein 109 cells−1) with an average specific activity of 3942 ± 765 pg PGE2 μg−1 microsomal protein 5 min−1.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Berglund P, Sjödberg M, Garoff H, Atkins JG, Sheahan BJ and Liljeström P (1993) Semliki Forest Virus expression system: production of infectious recombinant particles. Bio/Technology 11, 916–920.
Blasey HD, Isch C and Bernard AR (1995) Cellcube: a new system for large scale growth of adherent cells. Biotechnology Techniques 9,10, 727–730.
Liljeström P (1994) Alphavirus expression systems, current opinion in Biotechnology, 5, 495–500.
Liljeström P and Garoff H (1991a) A new generation of animal cell expression vectors based on the Semliki Forest Virus system. Bio/Technology 9, 1356–1361.
Liljeström and Garoff H (1991b) Current protocols. Mol. Biol. 2, 16–20.
Lundström K, Mills A, Buell G, Allet E, Adami N and Liljeström P (1994) High-level expression of the human neurokine-1 receptor in mammalian cell lines using the Semliki Forest Virus expression system. Eur. J. Biochem. 244, 917–921.
Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, Liou S, Simmons D and Hwang D (1992) Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J. Biol. Chem. 26, 25934–25938.
Lundström K, Vargas A and Allet B (1995) Functional activity of a biotinylated human neurokinin 1 receptor fusion expressed in the Semliki Forest Virus system. Biochem. Biophys. Res. Comm. 208,1, 260–266.
Mikus P, Urano T, Liljeström P and Ny T (1993) Plasminogen activator inhibitor type 2 (PAI-2) is a spontaneously polymerising SEPRIN: biochemical characterisation of the recombinant intracellular and extracellular form. Eur. J. Biochem. 218, 1071–1082.
Olkkonen VM, Liljeström P, Garoff H, Simons K and Dotti C (1993) Expression of heterologous proteins in cultured rat hippocampal neurons using the Semliki Forest Vector. J. Neurosci. Res. 35, 445–451.
Paul NL, Marsh M, McKeating JA, Schulz TF, Liljeström P, Garoff H and Weiss RA (1993) Expression of HIV-1 envelope glycoproteins by Semliki Forest Virus Vectors. AIDS Res. Hum. Retroviruses 9, 963–970.
Pritchard K, O'Banion MK, Miano J, Vlasic N, Bhatia U, Young D and Stemerman M (1994) Induction of Cyclooxygenase-2 in rat vascular smooth muscle cells in vitro and in vivo. J. Biol. Chem. 269, 8504–8509.
Xie W, Robertson DL and Simmons DL (1992) Mitogen-inducible prostaglandin G/H Synthase: a new target for nonsteroidal anti-inflammatory drugs. Drug. Dev. Res. 25, 249–265.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Blasey, H., Lundström, K., Tate, S. et al. Recombinant protein production using the Semliki Forest Virus expression system. Cytotechnology 24, 65–72 (1997). https://doi.org/10.1023/A:1007974121182
Issue Date:
DOI: https://doi.org/10.1023/A:1007974121182