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A novel synthetic mammalian promoter derived from an internal ribosome entry site
Author(s) -
Hartenbach Shizuka,
Fussenegger Martin
Publication year - 2006
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21174
Subject(s) - chinese hamster ovary cell , promoter , biology , transgene , microbiology and biotechnology , ribosomal binding site , internal ribosome entry site , gene , gene expression , messenger rna , cell culture , genetics , translation (biology)
of specific mutations into a synthetic internal ribosome entry site (IRES GTX ) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P GTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high‐level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF 121 ), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus ‐derived secreted α‐amylase (SAMY) in Chinese hamster ovary cells (CHO‐K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P GTX2 ) doubled the SEAP expression levels in CHO‐K1. P GTX2 reached near 70% of P SV40 ‐driven expression levels and outperformed constitutive phosphoglycerate kinase (P PGK ) and human ubiquitin C (P hUBC ) promoters in CHO‐K1. Also, P GTX2 was successfully engineered for macrolide‐inducible transgene expression. Owing to its size of only 182 bp, P GTX2 is one of the smallest eukaryotic promoters. Although P GTX2 was found to be a potent promoter, it retained its IRES GTX ‐specific translation‐initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells. © 2006 Wiley Periodicals, Inc.