AVS 50th International Symposium
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuP

Paper BI-TuP15
Creation of Surface Macromolecular Docking Sites for the Reversible Immobilization of Proteins in Active Conformation and Controlled Orientation

Tuesday, November 4, 2003, 5:30 pm, Room Hall A-C

Session: Poster Session
Presenter: G. Zhen, Swiss Federal Institute of Technology, Switzerland
Authors: G. Zhen, Swiss Federal Institute of Technology, Switzerland
E. Zobeley, Swiss Federal Institute of Technology, Switzerland
V. Eggli, Swiss Federal Institute of Technology, Switzerland
R. Glockshuber, Swiss Federal Institute of Technology, Switzerland
M. Textor, Swiss Federal Institute of Technology, Switzerland
Correspondent: Click to Email
Our contribution describes the comparative performance of two docking site concepts for the immobilization of biomolecules in active conformation and controlled immobilization. They are based on biotin-(Strep)avidin and NTA-Ni@super 2+@-histag linkage techniques, in combination with polycationic, PEG-grafted, NTA(nitrilotriacetic acid) functionalized and biotinylated copolymers. Enzyme @beta@-lactamase served as the model protein for the verification of the linkage concept and for the investigation of the activity of the surface-immobilized protein and its dependence on the molecular orientation. Five different variants of @beta@-lactamase with single cysteine site-directed mutagenesis on the surface were engineered. Two platforms were tested in order to determine how the mechanical and dynamic properties of the interface influence the enzyme-orientation-dependent catalytic activity: polymeric interface (flexible chains, soft) and functionalized alkanethiol monolayers on gold (comparatively immobile, stiff). The @beta@-lactamase variants were biotinylated at free thiol-group with a cleavable biotinylation reagent allowing for controlled release of the surface-bound enzyme. The immobilization was achieved on niobium oxide surface coated with biotinylated Poly(L-lysine)-g-poly(ethylene glycol) and on gold coated with mixture of alkanethiol-biotin and alkanethiol-OH self-assembled monolayer. The biotinylated @beta@-lactamase was subsequently bound to the surface via NeutrAvidin. The long-term stability of the immobilized proteins was evaluated. The amount of immobilized @beta@-lactamase on the chips was measured by three different techniques: OWLS, SPR and specific enzymatic activity via photospectroscopic detection of the turnover of the chromogenic substrate nitrocefin. Specific immobilization could be discriminated from non-specific adsorption. Furthermore the effect of the immobilization scheme on the biological activity was quantitatively examined.