Construction and characterization of a specialized ribosome system for the overproduction of proteins in Escherichia coli.

A recombinant Escherichia coli system was constructed to overexpress proteins using the specialized ribosomes developed by Herman de Boer and co-workers at Genentech. Specialized ribosomes carry a mutation in the anti-Shine-Dalgarno region of 16 S ribosomal RNA, which is the site of messenger RNA binding. A complementary mutation in the ribosome binding site (the Shine-Dalgarno region) of a particular mRNA results in specific and efficient translation of this mRNA on the specialized ribosomes. Production of beta-galactosidase with this system was characterized with respect to transcription, translation, plasmid replication, and cell growth rate. Translation of specialized mRNA on specialized ribosomes gave 5 times more enzyme activity than did translation of wild-type mRNA on wild-type ribosomes under similar conditions. The system described here offers a number of advantages when compared to a similar system described recently: (1) two separate specialized systems were constructed; (ii) the genes for specialized mRNA and specialized rRNA are on separate plasmids, allowing for different combinations of mRNA and rRNA; and (iii) appropriate controls were constructed for each plasmid.