Engineering mRNA translation initiation to enhance transient gene expression in chinese hamster ovary cells.
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 Ser(51)Ala site-directed mutant of eIF2alpha, 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 eIF2alpha 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 eIF2alpha Ser(51)Ala 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 eIF2alpha protein had no such effect. Elevated luciferase activity was associated with a reduction in the level of eIF2alpha phosphorylation in cells transfected with the mutant eIF2alpha 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 Ser(51)Ala or wild-type eIF2alpha 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.
Underhill
Michèle F
MF
Research School of Biosciences, University of Kent at Canterbury, UK.
Coley
Clare
C
Birch
John R
JR
Findlay
Alison
A
Kallmeier
Robert
R
Proud
Christopher G
CG
James
David C
DC
D000818:Animals; D016466:CHO Cells / Q000378:metabolism* / Q000502:physiology; D006224:Cricetinae; D018274:Electroporation / Q000379:methods; D015852:Eukaryotic Initiation Factor-2 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000235:genetics*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D010957:Plasmids / Q000008:administration & dosage; D014176:Protein Biosynthesis / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection / Q000379:methods*
D015852:Eukaryotic Initiation Factor-2; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008156:Luciferases