Mislovicová D, Chudinová M, Gemeiner P, Docolomanský P
Institute of Chemistry, Slovak Academy of Sciences, Bratislava.
J Chromatogr B Biomed Appl. 1995 Feb 3;664(1):145-53. doi: 10.1016/0378-4347(94)00447-d.
This work presents confirmation of the biospecific character of the interaction of Concanavalin A (Con A) immobilized on bead cellulose with invertase. In spite of the extraordinary strong binding of invertase to this Con A conjugate (KD = 5 x 10(-9) M), conditions have been found to use Con A-bead cellulose as an affinity chromatography medium. The effective factor in the release of the bound invertase by the counter ligand (alpha-methyl-D-mannopyranoside) is the time of incubation. This phenomenon was demonstrated in both batch and flow-through experiments. A concentration of 1.5 mg Con A per ml of gel was found to be suitable with regard to the maximal invertase/Con A binding ratio and the optimal invertase recovery (94%). As a result of the strong biospecific interaction the purification of invertase was very effective (above ten-fold). Verification by FPLC and PAGE of the product purity revealed only one significant protein band.
这项工作证实了固定在珠状纤维素上的伴刀豆球蛋白A（Con A）与转化酶相互作用的生物特异性特征。尽管转化酶与这种Con A缀合物的结合异常牢固（解离常数KD = 5×10⁻⁹ M），但已发现一些条件可将Con A - 珠状纤维素用作亲和色谱介质。通过反配体（α-甲基-D-甘露吡喃糖苷）释放结合的转化酶的有效因素是孵育时间。这种现象在分批实验和流通实验中均得到了证明。发现每毫升凝胶中1.5毫克Con A的浓度对于最大转化酶/Con A结合率和最佳转化酶回收率（94%）而言是合适的。由于强烈的生物特异性相互作用，转化酶的纯化非常有效（超过十倍）。通过快速蛋白质液相色谱（FPLC）和聚丙烯酰胺凝胶电泳（PAGE）对产物纯度进行验证，结果显示只有一条明显的蛋白带。
