Identification and Characterization of a Novel Freshwater Mussel Adhesive Protein
Li, XiaoTong 1; Cameron, William 1; Ng, Judith 1; Rees, David 1, 4, 5; Rocheleau, Jonathan 1, 2, 3; Sone, Eli 1, 2, 3
1. IBBME, University of Toronto;
2. Faculty of Dentistry, University of Toronto;
3. Department of Materials Science & Engineering, University of Toronto;
4. University Health Network;
5. Department of Physiology
Nature-inspired bioadhesives have vast potential in medical applications and can overcome the challenges presented by adhesion in aqueous environments. The European freshwater mollusk Dreissena bugensis (quagga mussel) exhibits an exceptional ability to adhere to a wide range of surfaces underwater via the byssus. Over the last 30 years, marine mussels have been the most widely studied model of underwater adhesion. Their adhesive strength is directly proportional to 3,4-dihydroxyphenylalanine (Dopa) content within the foot protein; a post-translationally modified hydroxylated tyrosine residue. In contrast, quagga mussels are a structurally-similar but genetically-distinct freshwater mussel species that have very low amounts of Dopa in their foot proteins. The proteins responsible for quagga mussel adhesion are therefore unknown. In previous experiments observing the size and localization of Dreissena bugensis foot proteins (Dbfp), Dbfp7 was selected as the top adhesive protein candidate in quagga mussel adhesion. We propose that the adhesion mechanism adopted by quagga mussels function independently of Dopa, and we aim to elucidate the freshwater mussel adhesion mechanism with the long-term goal of providing a foundation towards the development of a novel bioadhesive.
The first aim of this project is to select the variants of Dbfp7 to produce through recombinant protein synthesis. This is achieved by determining the abundance and enrichment of Dbfp7 variants at the adhesive interface through LC-MS/MS. After variants of Dbfp7 are selected, we aim to express and purify recombinant Dbfp7 proteins in E. coli. Purified recombinant Dbfp7 proteins will be characterized with circular dichroism to predict secondary structure and surface adsorption kinetics can be measured through quartz crystal microbalance. Recombinant Dbfp7 will be compared to Dbfp7 proteins extracted natively from quagga mussels to observe differences in structure, function, as well as the importance of post translational modifications.
Dbfp7 variants 9 and 14 are determined to be the top adhesive candidates for quagga mussel adhesion. LC-MS/MS peptide localization analysis reveals the possibility of a post-translational cleavage in Dbfp7 proteins. To explore the adhesive capabilities of the cleaved and full-length versions of Dbfp7 proteins, vectors are arranged to make it possible for the expression of both full-length and cleaved versions of the protein. Expression of Dbfp7 proteins in E. coli appear to be non-toxic. High expression levels have been achieved and visualized using a GFP tag.
Bioadhesives inspired by quagga mussel adhesive proteins have the potential to be nontoxic to the human body, bind strongly in wet environments, and be biodegradable. Through this project, we can uncover the unknowns of quagga mussel adhesion, and therefore serve as a blueprint for the development of a novel bioadhesive used for medical and aqueous conditions.