Underhill, M.F. and Marchant, R.J. and Carden, M.J. and James, D.C. and Smales, C.M. (2006) On the effect of transient expression of mutated eIF2 alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock. Molecular Biotechnology, 34 (2). pp. 141-149. ISSN 1073-6085.
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There are a growing number of reports on the beneficial effects of subphysiological temperature in vitro culturing (27-35 degrees C) of mammalian cells on recombinant protein yield. However, this effect is not conserved across cell lines and target products, and our understanding of the molecular mechanism(s) responsible for increased recombinant protein yield upon reduced temperature culturing of mammalian cells is poor. What is known is that mammalian cells respond to cold-shock by attenuating global cap-dependent translation. Here, we have investigated the hypothesis that the cap-dependent attenuation of mRNA translation upon cold-stress of in vitro-cultured mammalian cells can be prevented, or at least alleviated, by overexpressing mutant translation initiation factors in Chinese hamster ovary and HeLa cells. We have shown that the transient coexpression of either an eIF2 alpha Ser(51)-> Ala(51) mutant or an eIF4ESer(209 ->)Glu(209) mutant with firefly luciferase affects luciferase expression levels in a cell line and temperature dependent manner. Further, regardless of the coexpression of initiation factors, transient reporter gene expression was enhanced at subphysiological temperatures(< 37 degrees C), suggesting that reduced temperature cultivation can be used to improve the yield of recombinant protein during transient expression. The implications of these results upon cell engineering strategies involving manipulation of the translational apparatus for the enhancement of recombinant protein synthesis upon cold-shock are discussed.
|Additional information:||Sp. Iss. SI|
|Divisions:||Faculties > Science Technology and Medical Studies > School of Biosciences|
|Depositing User:||Mark Smales|
|Date Deposited:||03 Sep 2008 06:56|
|Last Modified:||28 Mar 2012 10:51|
|Resource URI:||http://kar.kent.ac.uk/id/eprint/6223 (The current URI for this page, for reference purposes)|
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