On the effect of transient expression of mutated eIF2alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock.
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 eIF2alphaSer51 Ala51 mutant or an eIF4ESer209 Glu209 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.
Underhill
Michèle F
MF
Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Marchant
Rosalyn J
RJ
Carden
Martin J
MJ
James
David C
DC
Smales
C Mark
CM
D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D018929:Cell Culture Techniques; D003080:Cold Temperature; D006224:Cricetinae; D003412:Cricetulus; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics; D009154:Mutation; D014176:Protein Biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis
D015852:Eukaryotic Initiation Factor-2; D039561:Eukaryotic Initiation Factor-4E; D012333:RNA, Messenger; D011994:Recombinant Proteins; D049409:Luciferases, Firefly