期刊
MOLECULAR BIOTECHNOLOGY
卷 34, 期 2, 页码 141-149出版社
HUMANA PRESS INC
DOI: 10.1385/MB:34:2:141
关键词
cold-shock; translation initiation; CHO cells; initiation factors; recombinant protein
资金
- Biotechnology and Biological Sciences Research Council [BB/D009375/1] Funding Source: Medline
- Biotechnology and Biological Sciences Research Council [BB/D009375/1] Funding Source: researchfish
- BBSRC [BB/D009375/1] Funding Source: UKRI
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 eIF2 alpha Ser(51)-> Ala(51) mutant or an eIF4ESer(209 ->)Glu(209) 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.
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