A Method of Immobilization on the Solid Support of Complex and Simple Enzymes Retaining Their Activity

Immobilization of native proteins, retaining their activity, on the solid support is often crucial for a variety of biochemical assays involving protein–protein interactions. In this study we describe a technique which allows binding of both complex (protein kinase CK2) and simple (calf intestine alkaline phosphatase, CIP) enzymes to the solid support without denaturization of the proteins. This method is based on the covalent cross-linking of the enzymes to the bifunctional resin, containing the secondary amino and thiol groups, in a coupling reaction with the imidoester dimethyl pimelimidate hydrochloride. Both enzymes in their bound form were active in the specific biochemical assays. We also found that the CK2 and CIP resins did not change their activity for at least 3 months, and the quality of these resins were not affected by high salts or reducing agents. Thus, this method can be recommended for general use to generate active enzymes coupled to the solid support.