Recombinant Self-Assembling Peptides as Biomaterials for Tissue Engineering

Biomaterials. 2010 Dec;31(36):9395-405. doi: 10.1016/j.biomaterials.2010.08.051. Epub 2010 Oct 8.

Abstract

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. P₁₁-4 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 7.5 but at low pH adopts an antiparallel β-sheet conformation. It also self-assembles under physiological conditions in a concentration-dependent manner. Here we describe an unimer P₁₁-4 production system and the use of a simple site-directed mutagenesis approach to generate a series of other P₁₁-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. 4.64 g/L and we believe the highest reported recovery of a recombinant self-assembling peptide at 203 mg/L of pure recombinant P₁₁-4. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Biocompatible Materials / chemistry
  • Biocompatible Materials / pharmacology*
  • Cell Death / drug effects
  • Cloning, Molecular
  • Cyanogen Bromide / metabolism
  • Cytoplasm / drug effects
  • Cytoplasm / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli
  • Humans
  • Hydrogels / pharmacology
  • Inclusion Bodies / ultrastructure
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptides / chemistry
  • Peptides / isolation & purification
  • Peptides / metabolism
  • Protein Structure, Secondary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / pharmacology*
  • Solubility / drug effects
  • Tissue Engineering / methods*

Substances

  • Biocompatible Materials
  • Hydrogels
  • Peptides
  • Recombinant Fusion Proteins
  • Cyanogen Bromide