Site specific biotinylation of the human aldo/keto reductase AKR1A1 for immobilization

New strategies for the specific monolabelling of enzymes play a key role in the development of artificial proteins. Especially for the emerging research field of nanobiotechnology and bioelectronics artificial monofunctionalized redoxproteins are of great interest. The human AKR1A1, an enzyme of the aldo/keto reductase superfamily, has been chosen as subject for the synthesis of an artificially mono biotinylated redoxprotein in order to selectively immobilize this enzyme for bioelectronic applications. To produce monofunctionalized enzyme we applied the strategy of Expressed Protein Ligation (EPL) in combination with solid phase peptide synthesis (SPPS). Accordingly, we used the IMPACT®-system and cloned the aldo/keto-reductase as fusion protein with an additional intein/chitin binding domain. Through intein mediated splicing we could produce the C-terminal thioester of the aldo/keto-reductase, which maintained its biological activity. Then, the thioester was coupled to Cys-Lys(Ahx-Ahx-biotin)-amide by Native Chemical Ligation, which led to mono-biotinylated protein. The enzyme activity was proven to be intact as shown by various kinetic investigations. Immobilization was performed on avidin coated silica microspheres. Accordingly, for the first time selectively modified AKR1A1 has been immobilized.