Developing Polymeric Proteins for Cell Culture

Abstract

The possibility of using protein biomaterials as platforms to generate stimuli that can promote cell activity is a strategy that is receiving more attention. Nevertheless, the use of proteins isolated from animal sources for regenerative medicine is likely associated with a risk of infection and undesirable immune responses. This study used a polymeric bacterial surface protein, capsular antigen fraction 1 protein (Caf1), from the plague bacterium Yersinia pestis, which shares a 3D structure with the largest class of human extracellular matrix (ECM) proteins, including fibronectin. It has five surface loops that are suitable for inserting active peptides from ECM proteins to produce bioactive Caf1 variants. Different peptides from the ECM proteins varying in length and function were inserted into Caf1 protein. The Caf1 mutants were expressed and purified by biochemical methods and used to coat flat surfaces. Surfaces coated with Caf1 (labelled with the fluorescent dye, Fluorescein isothiocyanate (FITC)) were fluorescently scanned and the protein localised. Caf1 was also formed into 3D gels and mixed with alginate to create composite hydrogels. These were examined with confocal microscopy. Results showed that it was possible to insert peptides into different sites of the Caf1 polymer including the polymer termini. This controlled engineering of sites within the polymer allowed us to study the insertion of different length peptides into the polymer. The fluorescent intensity scanning protocol quantified Caf1 proteins adhesion capacity onto surfaces and showed their distribution and accumulation level which helped in optimal surface coating with proteins. Cell culture assays on surfaces coated with different concentrations (1000, 750, 500, 400, 250, 200, 100 µg/ml) Caf1 proteins showed the ability of some peptides cloned into Caf1 proteins to enhance human keratinocyte adhesion, support their growth, and accelerate their motility into a “wounded” area. Future studies should focus on incorporating mixed peptides into the Caf1 polymer to expand their cell activity window. They should also focus on improving Caf1 hydrogels in term of surfaces fabrication and biodegradability as well as their ability to form composite polymers with different materials. This could produce a wide range of materials that could be implemented in the field of regenerative medicine.