Increased synthesis in E. coli of fibroblast and leukocyte interferons through alterations in ribosome binding sites.
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.
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*
D007370:Interferon Type I; D012329:RNA, Bacterial; D012333:RNA, Messenger