Overexpression and purification of the trimeric aspartate transcarbamoylase from Bacillus subtilis.
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.
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
D P
DP
Baker
J M
JM
Aucoin
M K
MK
Williams
L A
LA
deMello
E R
ER
Kantrowitz
D001221:Aspartate Carbamoyltransferase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics*; D002221:Carbamyl Phosphate / Q000737:chemistry / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D010957:Plasmids / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D001224:Aspartic Acid; D002221:Carbamyl Phosphate; D001221:Aspartate Carbamoyltransferase