Marinkovic D V, Marinkovic J N
Prep Biochem. 1977;7(1):9-18. doi: 10.1080/00327487708062109.
The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of beta-galactosidase from Escherichia coli mutant M15. The crude extract of mutant M15 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-aminophenyl-beta-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as beta-galactosidase from E. coli.
亲和层析技术已用于从大肠杆菌突变体M15中对β-半乳糖苷酶的可互补组分和互补酶进行部分纯化。将突变体M15的粗提取物与片段CM-B一起孵育。互补酶和可互补组分通过已共价连接抑制剂的对氨基苯基-β-D-硫代半乳糖苷小柱。大部分非特异性蛋白质直接通过亲和柱，而特异性酶蛋白则保留与凝胶结合。用含氯化钠的磷酸盐缓冲液从柱上洗脱可互补组分。用硼酸钠缓冲液从亲和支持物上洗脱结合的互补酶。本研究结果表明，100%的互补酶与柱结合。部分纯化的酶在圆盘凝胶电泳中的位置与大肠杆菌的β-半乳糖苷酶相同。
