Increased Synthesis in E. coli of Fibroblast and Leukocyte Interferons Through Alterations in Ribosome Binding Sites

Authors:
  • HSH. Michael Shepard
    H. Michael Shepard
  • EYElizabeth Yelverton
    Elizabeth Yelverton
  • DGDavid V. Goeddel
    David V. Goeddel
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Abstract
Eleven chimeric plasmids have been constructed which direct the synthesis of mature human fibroblast (IFN-β1) or leukocyte interferon (IFN-αA) 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-αA varied about six-fold, while among the different IFN-β1 expression plasmids a range of more than 100-fold in interferon production was observed. The difference in the range of variation between the IFN-αA and IFN-β1 plasmids is attributed partly to changes in messenger RNA secondary structure within the ribosome binding sites which affect message half-life.

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