Protein engineering of Caf1 from the plague bacterium Yersinia pestis for tissue engineering applications

Institute of Cellular and Molecular Bioscience, Medical School, University of Newcastle, UK, 2 Department of Bioengineering, Faculty of Engineering, Karamanoglu-Mehmetbey University, Karaman, Turkey The capsular antigen F1 (Caf1) protein of Y. pestis forms a gel-like, non-stick coat, allowing the bacteria to resist phagocytosis by macrophages. As cells cannot adhere to Caf1, new functions can be engineered in to control cell adhesion, differentiation and proliferation, through the mutation of the protein at key sites. Previously, a mutant Caf1 polymer containing an insertion mutant corresponding to the integrin binding motif (RGD) was produced, which reversed the non-stick phenotype and facilitated the adhesion of cells. Caf1 can also be made to form a hydrogel, highlighting the potential for this protein in tissue engineering applications. Building on this work, we test Caf1’s ability to retain its thermostability under different chemical conditions, and demonstrate its resistance to common proteases. We then show that several regions of the protein can be modified to contain new functional mutations such as growth factor peptides, cell adhesion motifs and protease recognition sites which allow for specific polymer cleavage. Finally, we show the engineered proteins can be combined to form mixed Caf1 polymers with multiple properties, similar to extracellular matrix proteins. The production of defined Caf1 polymers with different functionalities will greatly expand its use as a material in regenerative medicine, for example as a wound care product