Overproduction and purification of the regulatory subunit of Escherichia coli aspartate transcarbamoylase

An Escherichia coli strain/plasmid system has been developedfor the overexpression of the regulatory subunit of E.coli aspartatetranscarbamoylase (ATCase). Production of large quantities ofregulatory subunit, by the method described here, should facilitatefuture experiments, such as X-ray crystallography, NMR and hybridizationexperiments, aimed at understanding the heterotropic mechanismthat regulates the activity of ATCase. The plasmid used forthe over-expression carries the gene for the regulatory subunit,pyrI, downstream from the strong pyrB promoter. The host strain,EK1104 [Nowlan, S.F. and Kantrowitz, E.R. (1985) J. Biol. Chem.,260, 14712–14716] carries a deletion in the pyrBI regionof the chromosome, as well as a leaky pyrF allele. When thisstrain/plasmid system is grown under limiting pyrimidine levels,large quantities of the regulatory subunit of ATCase are producedwithout any trace of catalytic subunit or holo-enzyme. A procedurefor the purification of the regulatory subunit from cell extractshas also been developed yielding {small tilde}50 mg of purifiedregulatory subunit per liter of initial culture. The regulatorysubunit produced in this fashion is fully competent in reassociationexperiments with the native catalytic subunit. Furthermore,the reassociated holoenzyme exhibits kinetic properties identicalto those of the wild type enzyme. In addition, we report theconstruction of a pUC119 based plasmid which carries a uniqueNdeI site at the fMet of the pyrB gene of ATCase. This plasmid,which was used in the construction of the system for the overexpressionof the regulatory subunit of ATCase, has been shown to be ofgeneral use for the expression of foreign proteins in E.coli.