Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites.
1. Expression plasmids containing non-overlapping tandemly repeated ribosome binding sites (RBS) were constructed in order to stabilize mRNA and enhance translation. 2. Two synthetic genes (human calcitonin tetramer gene and a fusion gene human gamma-interferon-human calcitonin) were cloned in these vectors and the effect of multiplicity of Shine-Dalgarno (S/D) sequence on heterologous gene expression was studied. 3. It was found that duplication and triplication of RBS had no effect on the stability of mRNA but led to a strong decrease in the level of recombinant protein and mRNA in the cell. 4. Plasmids bearing four times repeated S/D sequences gave longer-lived mRNAs and maintained a level of protein and mRNA very close to the values obtained with a single S/D containing plasmids.
Alexciev
K
K
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
Uscheva
A
A
Pavlova
M
M
Yavachev
L
L
Ivanov
I
I
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002116:Calcitonin / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics*; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011863:Radioimmunoassay; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism*; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D012333:RNA, Messenger; D002116:Calcitonin