A Novel Method for the Covalent and Site Specific Immobilization of Proteins

Protein immobilization is an important step of bioengineering. The attachment of functional proteins onto a solid support or inside a polymer matrix is usually necessary to exploit the activity of the protein. However, proteins can easily denature and lose their function during these manipulation. Maintaining the protein structure, stability, and activity are the major challenges during the development of immobilization technology. Here we developed a method to covalently and site-specifically attach bioactive proteins through the metabolic incorporation of a non-natural amino acid azidohomoalaine (AHA). Due to its structural similarly to methonine, AHA can be used to replace methionine in protein synthesis. In the next step, AHA-containing protein can be covalently immobilized through its bio-orthogonal reaction to an alkyne group via the well-established click chemistry. Using a pyrophosphatase PpaC as the model protein, we demonstrated that the protein activity was not affected by the AHA incorporation.  However, since the location of methonine residues are usually inside the folded protein structure, the limited accessibility prevented efficient immobilization. Therefore, we designed and introduced a novel methionine tag at the C-terminus of the protein and shown that the tag could be used to attach PpaC to a supporting matrix via AHA incorporation and subsequent click chemistry reaction. We expect this method and the methionine tag to be generally useful to a broad range of proteins including membrane proteins. Our future plan include the application of this method into the construction of functional biomimetic membranes.