Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability

Abstract

An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.