title: Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: overproduction of human and murine cytokines.
abstract: We describe the construction, expression characteristics and some applications of a versatile dual-promoter expression plasmid for heterologous gene expression in Escherichia coli which contains both lambda pL and PT7 promoters. Furthermore, the plasmid is optimized to allow the expression of mature coding sequences without compromising the strength of the highly efficient PT7 or of the T7g10 ribosome-binding site. The effect of the the naturally occurring RNA loops at both the 5' and 3' ends of the T7g10 mRNA on expression was also examined. A double T7 RNA polymerase transcription terminator was inserted to ensure more reliable transcription termination and a higher expression level of the preceding gene. Further improvements involve a clockwise orientation of the promoters to minimize read-through transcription from plasmid promoters, a largely extended multiple cloning site, an antisense phage T3 promoter and a phage f1-derived, single-stranded replication origin. Variants of this plasmid allow for the production of fusion proteins with part of T7g10, a hexahistidine peptide and an enterokinase recognition site. The potential of these expression vectors is demonstrated by comparing the expression levels of a number of mammalian cytokines (human tumor necrosis factor, human immune interferon, human and murine interleukins 2, murine interleukin 4 and murine fibroblast interferon), using these expression plasmids.
authors.affiliation: Laboratory of Molecular Biology, University of Gent, Belgium.
authors.forename: N
authors.initials: N
authors.lastname: Mertens
authors.forename: E
authors.initials: E
authors.lastname: Remaut
authors.forename: W
authors.initials: W
authors.lastname: Fiers
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007372:Interferons / Q000096:biosynthesis / Q000235:genetics; D007378:Interleukins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins
chemical_list: D016207:Cytokines; D007378:Interleukins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D007372:Interferons; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases