Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria.
Mammalian genes, when inserted into bacterial plasmid or phage DNAs, will not be expressed into the corresponding specific proteins in E. coli unless proper initiations signals required for recognition by E. coli ribosomes are provided. We have studied these signals and chemically synthesized two DNA duplexes each containing different initiation signals. These have been inserted in front of the Simian virus 40 (SV40) small tumor antigen gene (SV40 t gene) at varying distances from the ATG initiation codon prior to its cloning into pBR322 plasmid DNA. Plasmid containing clones carrying either of these two synthetic ribosome binding sites (RBS) at varying distances from the SV40 t gene all produced a 17K protein identical to authentic t antigen by immunologic, electrophoretic and proteolytic digestion analyses. This provides a novel method to ensure the specific expression of any contiguous mammalian gene to be cloned to bacteria, and also a unique in vivo method for studying the structure-function (efficiency) relationship of RBS with specific base changes.
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D010957:Plasmids; D014176:Protein Biosynthesis; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism*; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic
D004279:DNA, Viral; D011948:Receptors, Antigen, T-Cell