Engineered Caf1 Protein Polymers form Tuneable Bioactive Hydrogel Scaffolds

Abstract

Here, we describe a new animal-free biomaterial with potential uses in 3D-tissue culture and regenerative medicine due to its low cost, high stability and definable bioactivity. Capsular antigen fraction1 (Caf1) is a protein from the plague bacterium Yersinia pestis that is secreted via the chaperone-usher pathway and protects the pathogen from phagocytosis by forming a non-stick protective layer around the cell. The 15.5kDa monomer has an Ig-like fold and resembles the extracellular matrix protein fibronectin. The subunits polymerise via donor-strand complementation, forming a highly stable non-covalent polymer. In this work, we demonstrate the production of recombinant Caf1 polymers via batch fermentation using Escherichia coli. These are secreted by the bacterium into a flocculent layer above the cell pellet, and can be easily extracted and purified in large quantities. We show the polymer retains its robust thermostability in a variety of chemical conditions by circular dichroism and SDS-PAGE, and observe the large size of polymers by electron microscopy and SEC-MALS. This analysis revealed polymers containing up to 250 subunits, with lengths >1.5 µm and weights in the MDa range. Additionally, we have selectively reversed the natural non-stick behaviour of the WT polymer by introducing an integrin binding sequence, RGDS, into loop5 that can promote fibroblast adhesion to the polymer surface. Additional bioactive peptides (up to 19 amino acids in length), including bone morphogenic protein2, collagen and laminin motifs, were then introduced at different positions within Caf1. Finally, PEG based chemical cross linkers were used to form stable 3D hydrogels with designed porosities and tuneable stiffness, ideal for use in cell culture and drug delivery applications. The combination of these motifs into tuneable Caf1 hydrogels will help expand the functionality of this exciting new biomaterial for use in a variety of biomedical applications.