Overproduction and purification of the regulatory subunit of Escherichia coli aspartate transcarbamoylase.
An Escherichia coli strain/plasmid system has been developed for the overexpression of the regulatory subunit of E. coli aspartate transcarbamoylase (ATCase). Production of large quantities of regulatory subunit, by the method described here, should facilitate future experiments, such as X-ray crystallography, NMR and hybridization experiments, aimed at understanding the heterotropic mechanism that regulates the activity of ATCase. The plasmid used for the 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 region of the chromosome, as well as a leaky pyrF allele. When this strain/plasmid system is grown under limiting pyrimidine levels, large quantities of the regulatory subunit of ATCase are produced without any trace of catalytic subunit or holoenzyme. A procedure for the purification of the regulatory subunit from cell extracts has also been developed yielding approximately 50 mg of purified regulatory subunit per liter of initial culture. The regulatory subunit produced in this fashion is fully competent in reassociation experiments with the native catalytic subunit. Furthermore, the reassociated holoenzyme exhibits kinetic properties identical to those of the wild type enzyme. In addition, we report the construction of a pUC119 based plasmid which carries a unique NdeI site at the fMet of the pyrB gene of ATCase. This plasmid, which was used in the construction of the system for the overexpression of the regulatory subunit of ATCase, has been shown to be of general use for the expression of foreign proteins in E. coli.
Dembowski
N J
NJ
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02167.
Kantrowitz
E R
ER
D000494:Allosteric Regulation; D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007700:Kinetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014498:Uracil / Q000494:pharmacology
D011994:Recombinant Proteins; D014498:Uracil; D001221:Aspartate Carbamoyltransferase; C037106:endodeoxyribonuclease NdeI; D015252:Deoxyribonucleases, Type II Site-Specific