Main > PROTEINS > Poly(Saccharide) Conjugates > Green Fluorescent Protein/Chitosan> > Org.: USA. U (BioSynthesis) > NPLS Contents
AUTHORS are looking into the use of enzymes to create functionally useful biopolymer-based materials that would be difficult to obtain by alternative synthetic routes.
"Our specific target has been to generate protein-polysaccharide conjugates". "In nature, protein-polysaccharide conjugates carry out important chemical, mechanical, and biological functions. Examples are the mucins that perform protective and lubricating functions for organs, and the proteoglycans, which are among the largest and most complex structures in mammalian cells and perform various mechanical and biological functions."
IN RECENT WORK, the group used tyrosinase to prepare conjugates of chitosan (a linear aminopolysaccharide) and green fluorescent protein (GFP). Tyrosinase is a copper-containing oxidative enzyme found in plant and animal tissues that catalyzes the production of melanin and other pigments.
"We mix the protein with chitosan and tyrosinase, and the conjugation occurs over a few hours". "We do not need protection and deprotection steps or reactive reagents, so the method can be performed in ONE POT without the need for special precautions or expensive reagents. We expect this method could be generalized to many proteins."
The method requires that the protein have accessible tyrosine residues for oxidation by the tyrosinase. Oxidation yields activated quinone residues that react with nucleophilic substituents of the chitosan.
"If a native protein lacks accessible tyrosine residues, we exploit molecular biological methods to genetically fuse a tyrosine-rich peptide sequence at one end of the protein". "Our results indicate that a tyrosine-rich fusion tail enhances tyrosinase-initiated protein-chitosan conjugation."
The group members showed that their GFP-chitosan conjugates can be deposited onto micropatterned gold electrodes in response to an applied voltage. "The deposition occurs with high spatial resolution, and GFP s fluorescence--and therefore structure--remains intact. "The incorporation of proteins into microfabricated devices offers considerable potential for biosensors and therapeutic devices. We are currently investigating this assembly procedure for applications in biosensors, microarrays, and microelectromechanical systems."
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