A bacteriophage transcription terminator permits the cloning of a mammalian expression vector carrying the human preprorenin gene in E. coli.
We constructed a plasmid-based expression vector carrying the murine metallothionein gene promoter, the human preprorenin gene, the Tn5 neomycin phosphotransferase II gene, and a complete bovine papilloma virus genome. We were unsuccessful in cloning the preprorenin gene in E. coli when it was inserted into a plasmid vector 3' to the metallothionein gene promoter. This result was consistent with our previous data suggesting that expression of the preprorenin gene is toxic to E. coli (Weighous, T.F. et al; (1986) Gene 45: 121-129). We then inserted a DNA fragment from bacteriophage lambda carrying the t1 transcriptional terminator between the promoter and the preprorenin gene; this vector was stably maintained in E. coli. Prior to introducing the vector DNA into mouse cells for expression studies the terminator was removed. Mouse cells carrying the vector DNA secreted high levels of human prorenin for greater than six weeks.
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D011994:Recombinant Proteins / Q000235:genetics*; D012083:Renin / Q000235:genetics*; D014158:Transcription, Genetic
D004279:DNA, Viral; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012083:Renin