The effects of hybrid ribosome-binding-site variants on the expression of human interferon-beta in Escherichia coli.
Human beta-interferon (IFN-beta) has been expressed in Escherichia coli by using vectors that join the trp promoter and a hybrid ribosome-binding site (HRBS) to the mature IFN-beta coding sequence. Introduction of an NcoI site at the ATG initiation codon of IFN-beta by site-directed mutagenesis has facilitated the construction of a series of portable HRBSs, by utilizing oligodeoxynucleotide adapters. The spacing between the Shine-Dalgarno (S-D) sequence and the initiator ATG ranged from 7-13 bp. One spacer of 9 bp length was varied at a single position. IFN-beta expression by these HRBS variants, as analyzed by antiviral activity and SDS-PAGE, shows both nucleotide sequence and spacer length effects. In vitro transcription-translation experiments indicate that some single base changes in the HRBS significantly decrease the rate of translation of the IFN-beta mRNA.
D003062:Codon; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
D003062:Codon; D007371:Interferon-gamma