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Expression of synthetic calcitonin genes in plasmid vectors containing tandemly repeated non-overlapping ribosome binding sites.

The International journal of biochemistry

Molecular Sequence Data, Binding Sites, genetics, Calcitonin, biosynthesis, Escherichia coli, Gene Expression, Genetic Vectors, Humans, Base Sequence, Nucleic Acid Conformation, Plasmids, Protein Biosynthesis, RNA, Bacterial, RNA, Messenger, Radioimmunoassay, Regulatory Sequences, Nucleic Acid, Repetitive Sequences, Nucleic Acid, Ribosomes, metabolism, Transcription, Genetic

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      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.

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