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Overexpression and Purification of the Trimeric Aspartate Transcarbamoylase From Bacillus Subtilis

Overexpression and Purification of the Trimeric Aspartate Transcarbamoylase From Bacillus Subtilis

D P Baker, J M Aucoin, M K Williams, L A deMello, E R Kantrowitz

Protein Expr Purif. 1995 Oct;6(5):679-84.

PMID: 8535162

Abstract:

A procedure has been developed for the overexpression and purification of milligram quantities of the Bacillus subtilis aspartate transcarbamoylase. The plasmid pEK171, carrying the B. subtilis pyrB structural gene under the control of the Escherichia coli pyrBI promoter, was transformed into the E. coli strain EK1104 and the enzyme overexpressed to approximately 50% of total soluble protein under extreme derepression of the pyrimidine pathway. The enzyme was subsequently purified by means of ammonium sulfate fractionation, anionic exchange chromatography using Q-Sepharose Fast Flow resin, negative chromatography on Matrex Gel Red A agarose, and hydrophobic interaction chromatography using Matrex Phenyl Cellufine. The purification yields approximately 60 mg of pure enzyme per liter of bacterial culture. Kinetic analysis of the overexpressed enzyme indicated that it had kinetic properties very similar to those of the enzyme purified from B. subtilis cells.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411027	Protein A-Agarose Fast Flow			Price

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