BIOT 285 |
| We have developed a generic strategy for the covalent assembly of proteins onto patterned surfaces, including sensor surfaces, by incorporating a tyrosine rich “pro-tag” at the C- or N-terminus of a protein of interest (Lewandowski et al., 2006). The tyrosyl residues of the pro-tag are enzymatically activated by tyrosinase to quinones, which then covalently couple to the primary amines of pH-stimuli responsive polysaccharide chitosan. The resulting protein-polysaccharide conjugate retains the pH-dependent solubility of chitosan, which enables its electrodeposition from aqueous solutions directly onto conductive polymers and electrode surfaces. Protein G has a specific binding affinity to the heavy chain constant (Fc) region of immunoglobulin G (IgG). Taking advantage of this feature, we fused a pentatyrosine pro-tag to the C-terminus of protein G, and then conjugated the fusion protein onto electrodeposited aminopolysaccharide chitosan. The assembled complex then retains affinity for IgG, including an anti-hexahistidine IgG. This forms the basis for a generic sensor for any recombinant protein product that contains the histidine-rich purification tag. Western blot and ELISA results confirm the expression and purification of proteinG-tyr and that the fusion protein retains its native activity in IgG binding. Spatially-directed immobilization of antibodies and his tag proteins on electrodeposited chitosan chips will be described. |
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Poster Session
5:30 PM-7:30 PM, Wednesday, August 22, 2007 BCEC -- Exhibit Hall - B2, Poster
Division of Biochemical Technology |