Engineering the Plague Capsular Antigen (Caf1) Protein: A Thermally Reformable Protein Polymer

Caf1 is a proteinaceous polymer produced by Yersinia pestis bacteria that helps them to evade phagocytosis. Individual 15 kDa Caf1 subunits associate non-covalently to form long (MDa, µm), highly thermostable polymers. Caf1 polymers can be combined with cross-linkers to form hydrogels of tuneable porosity and stiffness, are free from animal products, and are simple to produce and purify. Additionally, cells adhere poorly to the material, making it “non-stick”. Through mutagenesis, this phenotype can be reversed, allowing different bioactivities to be engineered into the protein. These properties make Caf1 a uniquely interesting biomaterial to develop for biomedical applications.

Here, we engineer Caf1 mutants containing signals involved in osteogenesis, and combine these to form a “mosaic” Caf1 polymer that can trigger the early stages of bone formation when used as a scaffold for bone marrow stromal cells. We next demonstrate that denatured Caf1 polymers can self-assemble into oligomers, including mosaic oligomers, allowing us to control the composition of bioactive subunits within the material. Finally, we show that we can use this property to create thermally reformable Caf1 hydrogels, providing a simple and effective route towards cell encapsulation within the 3D material. We anticipate that these developments will increase Caf1’s utility as a biomaterial for use in 3D cell culture, as well as other applications.