USING ANTISENSE MESSENGER RNA TO DOWNREGULATE LON MEDIATED PROTEOLYSIS IN ESCHERICHIA COLI
Bentley, William E
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The advent of metabolic engineering has instigated the introduction of foreign heterologous proteins into host cells, such as Escherichia coli. However, the metabolic burden incurred by the host cell to produce the desired recombinant protein elicits a cellular stress response that can result in reduced yields and degradation of the desired protein. In, lon is one of the major proteases responsible for this abnormal protein degradation, including recombinant proteins. Consequently, a variety of antisense strategies have been examined and shown to effectively control endogenous gene expression and function in E. coli. For this investigation we explored using a 300 base pair sequence of the 5' coding region of E. coli lon gene, including the start codon, cloned into both the pSE420 and pTO plasmids in the antisense (reverse) orientation. We examined the ability of lon antisense RNA to inhibit the production of endogenous lon protease and increase the protein yield and activity of a model recombinant protein, organophosphorus hydrolase (OPH). Results indicate that the overproduction of lon antisense did effectively downregulate the production of endogenous lon. In addition, cultures induced for lon antisense also revealed higher OPH protein levels in the first hour of production and a 7-fold higher activity.