Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.
A broad host-range expression plasmid was constructed comprising the incQ replicon, the recA promoter from Escherichia coli and the g10-L ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple "consensus" RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.
Monsanto Corporate Research, St. Louis, MO 63198.
S H
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Rangwala
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Fuchs
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DJ
Drahos
P O
PO
Olins
D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D015870:Gene Expression / Q000187:drug effects; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000235:genetics*; D013006:Growth Hormone / Q000235:genetics; D009268:Nalidixic Acid / Q000494:pharmacology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000235:genetics; D015183:Restriction Mapping; D001616:beta-Galactosidase / Q000235:genetics
D009268:Nalidixic Acid; D013006:Growth Hormone; D011938:Rec A Recombinases; D001616:beta-Galactosidase