title: Post-translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells.
abstract: Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37 degrees C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32 degrees C but was diminished at lower temperatures. The expression of both cap-dependent and cap-independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32 degrees C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post-translational events appear to proceed with higher fidelity and accuracy at 32 than 37 degrees C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32 degrees C recombinant cap-dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post-translational processing.
authors.affiliation: Protein Science Group, Department of Biosciences, University of Kent, Canterbury, Kent CT27NJ, UK.
authors.forename: Rosalyn J
authors.initials: RJ
authors.lastname: Masterton
authors.forename: Anne
authors.initials: A
authors.lastname: Roobol
authors.forename: Mohamed B
authors.initials: MB
authors.lastname: Al-Fageeh
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Carden
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D003080:Cold Temperature; D006224:Cricetinae; D003412:Cricetulus; D004587:Electrophoresis, Agar Gel; D008954:Models, Biological; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D011994:Recombinant Proteins