AVS 56th International Symposium & Exhibition
    Biomaterial Interfaces Tuesday Sessions
       Session BI-TuP

Paper BI-TuP5
Facile C-terminal Attachment of Proteins on Surfaces by Hydrazine-Intein Chemical Reactions

Tuesday, November 10, 2009, 6:00 pm, Room Hall 3

Session: Biomaterial Interfaces Poster Session I
Presenter: P. Yang, Duke University
Authors: P. Yang, Duke University
S. Marinakos, Duke University
A. Chilkoti, Duke University
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Protein immobilization on surfaces is important in many areas of research, including their biophysical characterization, affinity chromatography, and heterogeneous immunoassays. We describe a new site-specific C-terminal immobilization of proteins on surfaces to control the orientation and attachment point of the protein. Our strategy was to exploit intein chemistry and the related hydrazine attack reaction. To provide-proof-of principle of this approach, a tripartite thioredoxin-intein-elastin-like polypeptide (ELP) fusion was synthesized in E. coli from a plasmid-borne gene. In this fusion protein, the C-terminus of the target protein thioredoxin was fused with the N-terminus of an intein, a self-splicing protein domain. An ELP was fused to the C-terminus of the intein to facilitate purification of the fusion protein because ELP fusions allow simple purification of proteins from cell lysate by isothermal, salt-triggered phase transition of the ELP. Incorporation of the intein at the C-terminus of the target protein provided an unnatural peptide bond between the target protein and the intein, which is selectively reactive towards nucleophiles such as hydrazine. In order to immobilize the target protein –thioredoxin– a hydrazine-functionalized surface on a protein-resistant background was fabricated by modifying a gold-coated glass substrate with a mixed self-assembled monolayer consisting of a hydrazine-terminated hexa(ethylene glycol) thiol and a tri(ethylene glycol)thiol. We demonstrate that the hydrazine groups on the surface were able to directly attack the unnatural peptide bond between the target protein and the intein, liberating the thioredoxin from the tripartite fusion and covalently attaching it to the surface. As a result, the target protein was site-specifically immobilized on the planar substrates with uniform orientation. This technique provides many advantages over other site-specific immobilization methods, including faster reaction kinetics, higher surface density, and the ability to easily purify the protein prior to immobilization though a simple, one-step non-chromatographic process that exploits the phase transition of the ELP.