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.
Leipold
R J
RJ
Department of Chemical Engineering, University of Delaware, Newark 19716.
Dhurjati
P
P
D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012335:RNA, Ribosomal / Q000235:genetics; D012270:Ribosomes / Q000502:physiology*; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D011506:Proteins; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D007544:Isopropyl Thiogalactoside; D001616:beta-Galactosidase