Affinity Immobilization of Tagged Enzymes

The development of new enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native proteins could be used to create strong affinity bonds between the enzyme structure and a solid support functionalized with the complementary affinity ligand. The wide diversity of affinity systems creates many possibilities for enzyme immobilization. This work describes experimental strategies for affinity immobilization of tagged enzymes onto activated supports. Protocols for attaching histidine (His) and sugar-tagged enzymes onto common supports such as commercial aminated silica beads and activated graphite-cellulose are described in detail. Such strategies are generic and could be used in any application requiring immobilized biocatalysts, making future enzyme technologies more sensitive, simpler, re-usable, and less expensive. Finally, strategies for removing nonspecific adsorption and examples of possible applications of these methods to enzyme sensors are discussed.