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 p roteins using the specialized ribosomes developed by Herman de Boer an d co-workers at Genentech. Specialized ribosomes carry a mutation in t he anti-Shine-Dalgarno region of 16 S ribosomal RNA, which is the site of messenger RNA binding. A complementary mutation in the ribosome bi nding site (the Shine-Delgarno region) of a particular mRNA results in specific and efficient translation of this mRNA on the specialized ri bosomes. Production of beta-galactosidase with this system was charact erized 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 w ild-type mRNA on wild-type ribosomes under similar conditions. The sys tem described here offers a number of advantages when compared to a si milar 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 eac h plasmid.