Tuneable hydrogels of Caf1 protein fibers

Capsular antigen fraction 1 (Caf1) is a robust polymeric protein forming a protective layer around the bacterium Yersinia pestis. Occurring as ≈1 μm polymeric fibers, it shares its immunoglobulin-like fold with the majority of mammalian extracellular proteins such as fibronectin and this structural similarity suggests that this unusual polymer could form useful mimics of the extracellular matrix. Driven by the pressing need for reliable animal-free 3D cell culture environments, we showed previously that recombinant Caf1 produced in Escherichia coli can be engineered to include bioactive peptides, which influence cell behavior. Here, we demonstrate that through chemical crosslinking with a small palette of PEG-based crosslinkers, Caf1-based hydrogels can be prepared displaying a wide range of mechanical and morphological properties that were studied by rheology, compressive testing, SDS-PAGE and scanning electron microscopy. By varying the Caf1 protein concentration, viscoelasticity and stiffness (~11-2300 Pa) are reproducibly tunable to match natural and commercial 3D gels. Hydrogel porosity and swelling ratios were found to be defined by crosslinker architecture and concentration. Finally the hydrogels, which are 95-99% water, were shown to retain the high stability of the native Caf1 protein in a range of aqueous conditions, including extended immersion in cell culture media. The unusual Caf1 polymer thus offers the possibility of presenting bioactive protein subunits in a precisely tuneable hydrogel for use in cell culture and drug delivery applications.