Biomaterial Database

Superproduction and rapid purification of Escherichia coli aspartate transcarbamylase and its catalytic subunit under extreme derepression of the pyrimidine pathway. 1985

S F Nowlan, and E R Kantrowitz

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.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D009876	Operon	In bacteria, a group of metabolically related genes, with a common promoter, whose transcription into a single polycistronic MESSENGER RNA is under the control of an OPERATOR REGION.	Operons
D010957	Plasmids	Extrachromosomal, usually CIRCULAR DNA molecules that are self-replicating and transferable from one organism to another. They are found in a variety of bacterial, archaeal, fungal, algal, and plant species. They are used in GENETIC ENGINEERING as CLONING VECTORS.	Episomes,Episome,Plasmid
D011743	Pyrimidines	A family of 6-membered heterocyclic compounds occurring in nature in a wide variety of forms. They include several nucleic acid constituents (CYTOSINE; THYMINE; and URACIL) and form the basic structure of the barbiturates.
D004794	Enzyme Repression	The interference in synthesis of an enzyme due to the elevated level of an effector substance, usually a metabolite, whose presence would cause depression of the gene responsible for enzyme synthesis.	Repression, Enzyme
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D001221	Aspartate Carbamoyltransferase	An enzyme that catalyzes the conversion of carbamoyl phosphate and L-aspartate to yield orthophosphate and N-carbamoyl-L-aspartate. (From Enzyme Nomenclature, 1992) EC 2.1.3.2.	Aspartate Transcarbamylase,Co(II)-Aspartate Transcarbamoylase,Ni(II)-Aspartate Transcarbamoylase,Carbamoyltransferase, Aspartate,Transcarbamylase, Aspartate
D013045	Species Specificity	The restriction of a characteristic behavior, anatomical structure or physical system, such as immune response; metabolic response, or gene or gene variant to the members of one species. It refers to that property which differentiates one species from another but it is also used for phylogenetic levels higher or lower than the species.	Species Specificities,Specificities, Species,Specificity, Species
D014498	Uracil	One of four nucleotide bases in the nucleic acid RNA.
D046911	Macromolecular Substances	Compounds and molecular complexes that consist of very large numbers of atoms and are generally over 500 kDa in size. In biological systems macromolecular substances usually can be visualized using ELECTRON MICROSCOPY and are distinguished from ORGANELLES by the lack of a membrane structure.	Macromolecular Complexes,Macromolecular Compounds,Macromolecular Compounds and Complexes,Complexes, Macromolecular,Compounds, Macromolecular,Substances, Macromolecular