title: Increased synthesis in E. coli of fibroblast and leukocyte interferons through alterations in ribosome binding sites.
abstract: Eleven chimeric plasmids have been constructed which direct the synthesis of mature human fibroblast (IFN-beta 1) or leukocyte interferon (IFN-alpha A) proteins under the control of the E. coli trp promoter. The plasmids differ with respect to the nucleotide spacing between the Shine-Dalgarno sequence of the trp leader and the ATG translation start signal of the interferon genes. By utilizing a unique Xba I endonuclease site located within the spacer region of the expression plasmids, the spacings were altered from 2-10 nucleotides or 7-15 nucleotides for the fibroblast and leukocyte interferon expression plasmids, respectively. The optimal spacing for expression, as determined by interferon assay, is 9 nucleotides for both types of transcripts, despite differences in nucleotide sequence within the spacer region and downstream from the AUG initiator. Yields of IFN-alpha A varied about six-fold, while among the different IFN-beta 1 expression plasmids a range of more than 100-fold in interferon production was observed. The difference in the range of variation between the IFN-alpha A and IFN-beta 1 plasmids is attributed partly to changes in messenger RNA secondary structure within the ribosome binding sites which affect message half-life.
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005796:Genes; D006801:Humans; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D009690:Nucleic Acid Conformation; D010957:Plasmids; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D007370:Interferon Type I; D012329:RNA, Bacterial; D012333:RNA, Messenger