The construction of a synthetic Escherichia coli trp promoter and its use in the expression of a synthetic interferon gene.
An 82 base pair DNA fragment has been synthesised which contains the E. coli trp promoter and operator sequences and also encodes the first Shine Dalgarno sequence of the trp operon. This DNA fragment is flanked by EcoRI and ClaI/TaqI cohesive ends and is thus easy to clone, transfer between vector systems and couple to genes to drive their expression. It has been cloned into plasmid pAT153, producing a convenient trp promoter vector. We have also joined the fragment to a synthetic IFN-alpha 1 gene, using synthetic oligonucleotides to generate a completely natural, highly efficient bacterial translation initiation signal on the promoter proximal side of the IFN gene. Plasmids carrying this construction enable E. coli cells to express IFN-alpha 1 almost constitutively and with significantly higher efficiency than from a lacUV5 promoter based system.
D001482:Base Composition; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics*; D005813:Genes, Synthetic; D007372:Interferons / Q000235:genetics*; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics; D009876:Operon; D010957:Plasmids; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D013604:T-Phages / Q000201:enzymology
D009838:Oligodeoxyribonucleotides; D007372:Interferons; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004262:DNA Restriction Enzymes