Construction of a specialized-ribosome vector or cloned-gene expression in E. coli.
An expression system utilizing specialized ribosomes has been constructed with beta-galactosidase as the product. Ribosomes specific for lacZ mRNA are generated due to a mutation within the anti-Shine-Dalgarno region of a plasmidborne 16S rRNA gene that is complementary to a mutation within the ribosome-binding site of lacZ. Hence, a subpopulation of ribsomes specific for translation of the cloned gene mRNA is produced. Transcription of the lacZ gene is regulated by the tac promoter, while transcription of the mutated rrnB locus is controlled by the lambdaP(L) promoter. Batch experiments indicate that full induction of both operons (2 mM IPTG, 42 degrees C) leads to maximal beta-galactosidase activity per cell at levels 35% higher than that obtained using a wild-type ribosome expression system. Using a novel, site-directed mutagenesis technique, construction of the specialized ribosome vector is outlined, and the results of lacZ expression are presented as transcription of both the cloned-gene and the specialized-ribosome locus are induced.
Wood
T K
TK
Department of Chemical Engineering, North Carolina State University, Raleigh, North Carolina 27695-7905, USA.
Peretti
S W
SW