Baker D P, Aucoin J M, Williams M K, deMello L A, Kantrowitz E R
Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02167, USA.
Protein Expr Purif. 1995 Oct;6(5):679-84. doi: 10.1006/prep.1995.1089.
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.
已开发出一种用于过量表达和纯化毫克量枯草芽孢杆菌天冬氨酸转氨甲酰酶的方法。携带在大肠杆菌pyrBI启动子控制下的枯草芽孢杆菌pyrB结构基因的质粒pEK171被转化到大肠杆菌菌株EK1104中，并且在嘧啶途径的极端去阻遏条件下，该酶过量表达至约占总可溶性蛋白的50%。随后通过硫酸铵分级分离、使用Q-Sepharose Fast Flow树脂的阴离子交换色谱、在Matrex Gel Red A琼脂糖上的阴性色谱以及使用Matrex Phenyl Cellufine的疏水相互作用色谱对该酶进行纯化。每升细菌培养物的纯化产率约为60毫克纯酶。对过量表达的酶的动力学分析表明，其动力学性质与从枯草芽孢杆菌细胞中纯化的酶非常相似。
