Engineering mRNA Translation Initiation to Enhance Transient Gene Expression in Chinese Hamster Ovary Cells
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- 作者：Michè ; le F. Underhill ; Clare Coley ; John R. Birch ; Alison Findlay ; Robert Kallmeier ; Christopher G. Proud ; and David C. James
- 刊名：Biotechnology Progress
- 出版年：2003
- 出版时间：February 2003
- 年：2003
- 卷：19
- 期：1
- 页码：121 - 129
- 全文大小：252K
- 年卷期：v.19,no.1(February 2003)
- ISSN：1520-6033
文摘
To increase transient expression of recombinant proteins in Chinese hamster ovarycells, we have engineered their protein synthetic capacity by directed manipulationof mRNA translation initiation. To control this process we constructed a nonphosphorylatable Ser51Ala site-directed mutant of eIF2
, a subunit of the trimeric eIF2 complexthat is implicated in regulation of the global rate of mRNA translation initiation ineukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activityand 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 thattransient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted ina 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 luciferaseactivity was associated with a reduction in the level of eIF2 phosphorylation in cellstransfected with the mutant eIF2 construct. Transfection of CHO cells with theluciferase-only construct resulted in a marked decrease in the global rate of proteinsynthesis in the whole cell population 6 h post-transfection. However, expression ofthe mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesisin transfected cells to a level equivalent to or exceeding that of control cells. Associatedwith this, entry of plasmid DNA into cells during electroporation was visualized byconfocal microscopy using a rhodamine-labeled plasmid construct expressing greenfluorescent 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 cellitself functions to inhibit protein synthesis by signaling mechanisms affecting controlof mRNA translation by eIF2. This work therefore forms the basis of a rational strategyto generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.
, a subunit of the trimeric eIF2 complexthat is implicated in regulation of the global rate of mRNA translation initiation ineukaryotic cells. Phosphorylation of eIF2 by protein kinases inhibits eIF2 activityand 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 thattransient expression of the eIF2 Ser51Ala mutant with firefly luciferase resulted ina 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 luciferaseactivity was associated with a reduction in the level of eIF2 phosphorylation in cellstransfected with the mutant eIF2 construct. Transfection of CHO cells with theluciferase-only construct resulted in a marked decrease in the global rate of proteinsynthesis in the whole cell population 6 h post-transfection. However, expression ofthe mutant Ser51Ala or wild-type eIF2 proteins restored the rate of protein synthesisin transfected cells to a level equivalent to or exceeding that of control cells. Associatedwith this, entry of plasmid DNA into cells during electroporation was visualized byconfocal microscopy using a rhodamine-labeled plasmid construct expressing greenfluorescent 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 cellitself functions to inhibit protein synthesis by signaling mechanisms affecting controlof mRNA translation by eIF2. This work therefore forms the basis of a rational strategyto generically up-regulate transient expression of recombinant proteins by simultaneous host cell engineering.