Superproduction and rapid purification of Escherichia coli aspartate transcarbamylase and its catalytic subunit under extreme derepression of the pyrimidine pathway

• Published in: The Journal of biological chemistry 1985-11, Vol.260 (27), p.14712-14716
• Main Authors: Nowlan, S F, Kantrowitz, E R
• Format: Article
• Language: English
• Subjects: Aspartate Carbamoyltransferase - biosynthesis; Aspartate Carbamoyltransferase - genetics; Aspartate Carbamoyltransferase - isolation & purification; Bacteriology; Biological and medical sciences; Biotechnology; Enzyme Repression; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Genetic technics; Genetics; Kinetics; Macromolecular Substances; Methods. Procedures. Technologies; Microbiology; Mutant screening; Operon; Plasmids; Prokaryotes; Pyrimidines - biosynthesis; Species Specificity; Uracil - pharmacology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)38630-1

A strain of Escherichia coli has been constructed which greatly overproduces the enzyme aspartate transcarbamylase. This strain has a deletion in the pyrB region of the chromosome and also carries a leaky mutation in pyrF. Although this strain is a pyrimidine auxotroph, it will grow very slowly without pyrimidines if a plasmid containing the pyrB gene is introduced into it. Derepression occurs when this strain exhausts its uracil supply during exponential growth. Under extreme derepression, aspartate transcarbamylase can account for as much as 60% of the total cellular protein. This host strain/plasmid system can be utilized for the rapid purification of wild-type aspartate transcarbamylase or plasmid-born mutant versions of the enzyme. This system is particularly well-suited for analysis of the latter since the control of overproduction resides exclusively on the bacterial chromosome. Therefore, any plasmid bearing the pyrBI operon can be made to overproduce aspartate transcarbamylase in this host strain. Based on this system, a rapid purification procedure has been developed for E. coli aspartate transcarbamylase. The purification scheme involves an ammonium sulfate fractionation followed by a single precipitation of the enzyme at its isoelectric point. In a similar fashion, this strain can also be employed to produce exclusively the catalytic subunit of the enzyme if the plasmid only carries the pyrB gene. This system may be adapted to overproduce other proteins as well by using this host strain and the strong pyrB promoter linked to another gene.