Construction of a Specialized-Ribosome Vector for Cloned-Gene Expression in

An expression system utilizing specialized ribosomes has been constructed with p-galactosidase a s the product. Ribosomes specific for lacZ mRNA are generated due to a mutation within the anti-Shine-Dalgarno region of a plasmid-borne 16s rRNA gene that is complementary to a mutation within the ribosome-binding site of IacZ. Hence, a subpopu-lation of ribosomes specific for translation of the cloned-gene mRNA is produced. Transcription of the lacZ gene is regulated by the rac promoter, while transcription of the mutated rrnB locus is controlled by the hpr promoter. Batch experiments indicate that full induction of both operons (2 mM IPTG, 42°C) leads to maximal p-galactosidase activ-ity 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 spe-cialized ribosome vector is outlined, and the results of lacZ expression are presented a s transcription of both the cloned-gene and the specialized-ribosome locus are induced. Key words: ribosome vector cloned-gene expression Escherichia col