Modulation of translation‐initiation in CHO‐K1 cells by rapamycin‐induced heterodimerization of engineered eIF4G fusion proteins

Translation‐initiation is a predominant checkpoint in mammalian cells which controls protein synthesis and fine‐tunes the flow of information from gene to protein. In eukaryotes, translation‐initiation is typically initiated at a 7‐methyl‐guanylic acid cap posttranscriptionally linked to the 5′ end of mRNAs. Alternative cap‐independent translation‐initiation involves 5′ untranslated regions (UTR) known as internal ribosome entry sites, which adopt a particular secondary structure. Translation‐initiating ribosome assembly at cap or IRES elements is mediated by a multiprotein complex of which the initiation factor 4F (eIF4F) consisting of eIF4A (helicase), eIF4E (cap‐binding protein), and eIF4G is a major constituent. eIF4G is a key target of picornaviral protease 2A, which cleaves this initiation factor into eIF4G Δ and Δ eIF4G to redirect the cellular translation machinery exclusively to its own IRES‐containing transcripts. We have designed a novel translation control system (TCS) for conditional as well as adjustable translation of cap‐ and IRES‐dependent transgene mRNAs in mammalian cells. eIF4G Δ and Δ eIF4G were fused C‐ and N‐terminally to the FK506‐binding protein (FKBP) and the FKBP‐rapamycin‐binding domain (FRB) of the human FKBP‐rapamycin‐associated protein (FRAP), respectively. Rapamycin‐induced heterodimerization of eIF4G Δ ‐FKBP and FRB‐ Δ eIF4G fusion proteins reconstituted a functional chimeric elongation factor 4G in a dose‐dependent manner. Rigorous quantitative expression analysis of cap‐ and IRES‐dependent SEAP ‐ (human placental secreted alkaline phosphatase) and luc ‐ ( Photinus pyralis luciferase) encoding reporter constructs confirmed adjustable translation control and revealed increased production of desired proteins in response to dimerization‐induced heterologous eIF4G in Chinese hamster ovary (CHO‐K1) cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 210–225, 2003.