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Construction and Characterization of a Specialized Ribosome System for the Overproduction of Proteins in Escherichia coli
Author(s) -
Leipold Robert J.,
Dhurjati Prasad
Publication year - 1993
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp00022a001
Subject(s) - ribosome , ribosomal binding site , messenger rna , biology , ribosomal rna , translation (biology) , overproduction , escherichia coli , plasmid , protein biosynthesis , eukaryotic ribosome , rna , eukaryotic translation , shine dalgarno sequence , microbiology and biotechnology , transcription (linguistics) , genetics , gene , linguistics , philosophy
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‐Delgarno region) of a particular mRNA results in specific and efficient translation of this mRNA on the specialized ribosomes. Production of β‐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.