Staben C, Whitehead T R, Rabinowitz J C
Anal Biochem. 1987 Apr;162(1):257-64. doi: 10.1016/0003-2697(87)90035-2.
Rapid and convenient purification procedures based upon heparin-agarose chromatography for C1-tetrahydrofolate synthase from Saccharomyces cerevisiae and 10-formyltetrahydrofolate synthetase from Clostridium acidi-urici have been developed. The purification of the yeast enzyme involves three chromatographic steps that can be done rapidly, with no intervening dialyses, and results in high yield. The first step alone, heparin-agarose chromatography, is sufficient to purify the enzyme from yeast bearing a cloned copy of the ADE3 gene that overexpresses the protein. The other steps in the purification from wild-type yeast are matrex gel red A and phenyl-Sepharose chromatography. The purification of the clostridial enzyme involves protamine sulfate fractionation and heparin-agarose chromatography. Heparin-agarose also binds two other enzymes that use tetrahydrofolate, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase. Thus, heparin-agarose should prove useful in purification of a variety of enzymes that utilize tetrahydrofolate or its derivatives as a cofactor.
已开发出基于肝素 - 琼脂糖色谱法的快速便捷的纯化程序，用于从酿酒酵母中纯化C1 - 四氢叶酸合酶以及从尿酸梭菌中纯化10 - 甲酰四氢叶酸合酶。酵母酶的纯化涉及三个色谱步骤，这些步骤可以快速完成，无需中间透析，并且产率很高。仅第一步，即肝素 - 琼脂糖色谱法，就足以从携带过表达该蛋白的ADE3基因克隆拷贝的酵母中纯化该酶。从野生型酵母中纯化的其他步骤是基质凝胶红A和苯基 - 琼脂糖色谱法。梭菌酶的纯化涉及硫酸鱼精蛋白分级分离和肝素 - 琼脂糖色谱法。肝素 - 琼脂糖还结合另外两种使用四氢叶酸的酶，5,10 - 亚甲基四氢叶酸环水解酶和5,10 - 亚甲基四氢叶酸脱氢酶。因此，肝素 - 琼脂糖在纯化多种利用四氢叶酸或其衍生物作为辅因子的酶方面应证明是有用的。
