Recombinant self-assembling peptides as biomaterials for tissue engineering

Synthetic nanostructures based on self-assembling systems that aim to mimic natural extracellular matrix are now being used as substrates in tissue engineering applications Peptides are excellent starting materials for the self-assembly process as they can be readily synthesised both chemically and biologically P-11-4 is an 11 amino acid peptide that undergoes triggered self-assembly to form a self-supporting hydrogel It exists as unimers of random coil conformations in water above pH 75 but at low pH adopts an antiparallel beta-sheet conformation It also self-assembles under physiological conditions in a concentration-dependent manner Here we describe an unimer P-11-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P-11-family peptide expression vectors We have developed an efficient purification strategy for these peptide biomaterials using a simple procedure involving chemical cleavage with cyanogen bromide then repeated filtration lyophilisation and wash steps We report peptide-fusion protein yields of ca 464 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P-11-4 This peptide forms a self-supporting hydrogel under physiological conditions with essentially identical physico-chemical properties to the chemically synthesised peptide Critically it also displays excellent cytocompatibility when tested with primary human dermal fibroblasts This study demonstrates that high levels of a series of recombinant self-assembling peptides can be purified using a simple process for applications as scaffolds in tissue engineering (C) 2010 Elsevier Ltd All rights reserved