Lishko V K, Lishko O V, Hoffman R M
AntiCancer, Inc., San Diego, California 92110.
Protein Expr Purif. 1993 Dec;4(6):529-33. doi: 10.1006/prep.1993.1069.
Many types of human and animal tumors have an absolute requirement for methionine. This requirement can be satisfied by homocysteine only in normal cells and tissues. Therefore, methionine may be an important target in cancer therapy. To attack this target we have purified endotoxin-free methioninase from Pseudomonas putida by a novel and simple procedure. This procedure involves (1) a heat step of the cell extract at 60 degrees C for 8 min, (2) DEAE-Toyopearl ion-exchange chromatography, (3) DEAE-Sephadex A50 ion-exchange gel filtration chromatography, and (4) affinity chromatography on Acticlean to remove the endotoxin bound to the enzyme. The yield for this purification was up to 80%. The methioninase has four subunits of approximate molecular weight 43 kDa. This is the first methodology for methioninase that allows rapid purification with high yield and separation from endotoxin suitable for in vivo efficacy testing against methionine-dependent tumors in animal models.
许多类型的人类和动物肿瘤对甲硫氨酸有绝对需求。只有在正常细胞和组织中，同型半胱氨酸才能满足这种需求。因此，甲硫氨酸可能是癌症治疗的一个重要靶点。为了针对这个靶点，我们通过一种新颖且简单的方法从恶臭假单胞菌中纯化出了无内毒素的甲硫氨酸酶。该方法包括：（1）将细胞提取物在60℃加热8分钟；（2）DEAE - 托普雷斯离子交换色谱法；（3）DEAE - 葡聚糖A50离子交换凝胶过滤色谱法；（4）在Acticlean上进行亲和色谱以去除与酶结合的内毒素。这种纯化方法的产率高达80%。该甲硫氨酸酶有四个亚基，分子量约为43 kDa。这是第一种用于甲硫氨酸酶的方法，能够实现高产率的快速纯化，并与内毒素分离，适用于在动物模型中针对依赖甲硫氨酸的肿瘤进行体内疗效测试。
