Facile covalent bio-conjugation of hydroxyapatite

Hydroxyapatite, which is a major component of the bone system in the human body, is an attractive material for bio-applications because it is nontoxic as well as biocompatible. In addition, many proteins are readily adsorbed on the surface of hydroxyapatite. The protein adsorption properties of hydroxyapatite have been employed for many bio-applications such as protein purification and nanomedical application. Nevertheless, the applications are not appropriate for proteins possessing hydrophobic surfaces because the surface of hydroxyapatite is charged. For applications with hydrophobic proteins, a covalent conjugation would be an alternative approach instead of physical adsorption. However, only a few examples of the covalent conjugation of proteins onto the surface of hydroxyapatite have been reported due to the lack of a convenient method for covalent conjugation. Herein, we report a facile process for the covalent conjugation of proteins on hydroxyapatite. We have successfully activated the surface of hydroxyapatite by a simple treatment with a peptide coupling reagent (for instance, N,N′-dicyclohexyl carbodiimide, N,N′-diisopropylcarbodiimide, or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide). Then, we directly conjugated enhanced green fluorescent protein (EGFP) as a model protein, and the conjugation was confirmed by a fluorescent microscope. We also employed the method for a hydrophobic surface protein, lipase. In this case, we found that a linker compound is required for the conjugation of lipase because of the distinct polarities of the surfaces of hydroxyapatite and lipase. The conjugated lipase on hydroxyapatite exhibited higher activity in organic solvents than the free form of lipase by a factor of up to 30 and can be recycled without a significant loss of the activity.