Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells

Underhill, M. F., Coley, C., Birch, J. R., Findlay, A., Kallmeier, R., Proud, C. G. and James, D. C. (2003) Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells. Biotechnology Progress, 19 1: 121-129. doi:10.1021/bp025560b

Author Underhill, M. F.
Coley, C.
Birch, J. R.
Findlay, A.
Kallmeier, R.
Proud, C. G.
James, D. C.
Title Engineering mRNA translation initiation to enhance transient gene expression in Chinese hamster ovary cells
Journal name Biotechnology Progress   Check publisher's open access policy
ISSN 1097-0207
Publication date 2003-01
Sub-type Article (original research)
DOI 10.1021/bp025560b
Volume 19
Issue 1
Start page 121
End page 129
Total pages 9
Place of publication United States
Publisher American Institute of Chemical Engineers (AIChE)
Collection year 2003
Language eng
Subject C1
290600 Chemical Engineering
780105 Biological sciences
06 Biological Sciences
09 Engineering
10 Technology
Abstract To increase transient expression of recombinant proteins in Chinese hamster ovary cells, we have engineered their protein synthetic capacity by directed manipulation of mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site-directed mutant of eIF2, a subunit of the trimeric eIF2 complex that is implicated in regulation of the global rate of mRNA translation initiation in eukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activity and is known to increase as cells perceive a range of stress conditions. Using single-and dual-gene plasmids introduced into CHO cells by electroporation, we found that transient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted in a 3-fold increase in reporter activity, relative to cells transfected with reporter only. This effect was maintained in transfected cells for at least 48 h after transfection. Expression of the wild-type eIF2 protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2 phosphorylation in cells transfected with the mutant eIF2 construct. Transfection of CHO cells with the luciferase-only construct resulted in a marked decrease in the global rate of protein synthesis in the whole cell population 6 h post-transfection. However, expression of the mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesis in transfected cells to a level equivalent to or exceeding that of control cells. Associated with this, entry of plasmid DNA into cells during electroporation was visualized by confocal microscopy using a rhodamine-labeled plasmid construct expressing green fluorescent protein. Six hours after transfection, plasmid DNA was present in all cells, albeit to a variable extent. These data suggest that entry of naked DNA into the cell itself functions to inhibit protein synthesis by signaling mechanisms affecting control of mRNA translation by eIF2. This work therefore forms the basis of a rational strategy to generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.
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Created: Wed, 15 Aug 2007, 02:53:15 EST