Regulated protein production using site-specific recombination

Abstract

Abstract In vivo regulation of protein production is achieved by rearranging DNA segments comprising a protein-producing gene (i.e., protein-encoding DNA as well as regulatory DNA to effect the expression of the protein-encoding DNA in the host), in response to a change in an environmental condition such as temperature. The rearrangement is synchronized and directional (irreversible) in members of the cell population, because it is catalyzed by a lambda phage site-specific recombination enzyme system that operates on a pair of lambda phage attachment sites to rapidly drive the rearrangement and to avoid the reverse reaction. The cells include means to produce the lambda enzyme system in response to the change in environmental condition. By engineering one of the attachment sites within the gene that produces the protein whose production is to be regulated (yielding two gene segments), the synchronized rearrangement operates to change the gene from one configuration to another. In only one of these configurations are the gene segments positioned and oriented for protein production. Specifically, the protein-producing configuration is: gene segment one-attachment site-gene segment two. The attachment site is exogeneous to the gene, i.e, it does not occur in that location naturally and is positioned there by engineering techniques. The regulated protein production is useful, e.g., in fermenting the desired product, by allowing cell growth to proceed in the absence of product formation. When the desired cell mass is achieved, product production is enabled by the rearrangement.

Inventors

• 👤 Keith C. Backman

Bibliographic Details