Mateo C, Fernández-Lorente G, Cortés E, Garcia J L, Fernández-Lafuente R, Guisan J M
Departamento de Biocatálisis, Instituto de Catálisis, CSIC, Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Biotechnol Bioeng. 2001 Nov;76(3):269-76. doi: 10.1002/bit.10019.
Epoxy supports covalently immobilize proteins following a two-step mechanism; that is, the protein is physically adsorbed and then the covalent reaction takes place. This mechanism has been exploited to combine the selectivity of metal chelate affinity chromatography with the covalent immobilization capacity of epoxy supports. In this way, it has been possible to accomplish, in a simple manner, the purification, immobilization, and stabilization of a poly-His-tagged protein. To fulfill this objective we developed a new kind of multifunctional epoxy support (chelate epoxy support [CES]), which was tested using a poly-His-tagged glutaryl acylase as a model protein (an alphabeta-heterodimeric enzyme of significant industrial interest). The selectivity of the immobilization in CES toward poly-His-tagged proteins was dependent to a large extent on the density and nature of the chelated metal. The highest selectivity was achieved by using low-density chelate groups (e.g., 5 micromol/g) and metals with a low affinity (e.g., Co). However, the rate of covalent immobilization of the protein by its reaction with the epoxy groups on the support significantly increased at alkaline pH values. The multipoint attachment to the CES also depended on the reaction time. The immobilization of both glutaryl acylase subunits was achieved by incubation of the enzyme derivative at pH 10 for 24 h, with the best enzyme derivative 100-fold more stable than the soluble enzyme. By taking advantage of the selectivity properties of the novel support, we were able to immobilize up to 30 mg of protein per gram of modified Eupergit 250 using either pure enzyme or a very crude enzyme extract.
环氧载体通过两步机制共价固定蛋白质；也就是说，蛋白质先被物理吸附，然后发生共价反应。这种机制已被用于将金属螯合亲和色谱的选择性与环氧载体的共价固定能力相结合。通过这种方式，能够以简单的方式完成多聚组氨酸标签蛋白的纯化、固定和稳定化。为实现这一目标，我们开发了一种新型多功能环氧载体（螯合环氧载体[CES]），并使用多聚组氨酸标签的戊二酰基转移酶作为模型蛋白（一种具有重大工业意义的αβ异二聚体酶）对其进行了测试。CES对多聚组氨酸标签蛋白的固定选择性在很大程度上取决于螯合金属的密度和性质。通过使用低密度螯合基团（例如5微摩尔/克）和低亲和力的金属（例如钴）可实现最高的选择性。然而，在碱性pH值下，蛋白质与载体上环氧基团反应的共价固定速率显著增加。与CES的多点连接也取决于反应时间。通过在pH 10下将酶衍生物孵育24小时，实现了戊二酰基转移酶两个亚基的固定，最佳的酶衍生物比可溶性酶稳定100倍。利用新型载体的选择性特性，我们能够使用纯酶或非常粗的酶提取物，每克改性Eupergit 250固定多达30毫克的蛋白质。
