IGEM:IMPERIAL/2008/Bioprinter/Subteam 3

Biomaterials

We aim to synthesize two types of biomaterials, elastin and EAK16-II 3D scaffolds.

Elastin

Construct EP20-24-24 for human elastin polypeptide

Elastin is a polymeric extracellular matrix protein found in tissues requiring extensibility and elastic recoil, including arteries, lungs, ligaments and skin. The precursor for elastin is a soluble protein called tropoelastin. The sequence of tropoelastin consists of alternating hydrophobic and crosslinking domains, with distinct exons coding each domain.

Hydrophobic domains are rich in glycine, valine, proline and other non-polar amino acids, often present in tandem peptide repeats. Crosslinking domains are rich in alanine and contain lysine residues, which form covalent crosslinks that stabilise the polymeric form of elastin.

Under appropriate conditions of temperature and ionic strength, elastin undergoes a self-aggregation process called coacervation, in which the protein separate from solution as a second phase. Coacervation is usually induced by an increase in temperature. Unlike most proteins which undergo denaturation with increased temperature in solution, elastin polypeptides become more ordered through coacervation. The temperature at which coacervation occurs is dependent on ionic strength, pH, protein concentration and relative proportions of hydrophobic and hydrophilic residues in synthetic polypeptides.

EAK16-II

Molecular structure of EAK16-II peptide

EAK16-II is a self-assembling peptide, which forms stable β-sheet structures in water. It was found as a segment in zuotin, a yeast protein. The amino acid sequence for EAK16-II is AEAEAKAKAEAEAKAK. The molecular mass is estimated to be 1615.8.

The alternating positive and negative charges (--++--++) are responsible for electrostatic attraction between adjacent peptides. Self-assembly of EAK16-II peptide starts when the solution is exposed to physiological media or salt solution. When examined under SEM, a well-ordered nanofibre structure is observed. Nanofibres can futher aggregate to form a membranous 3D scaffold.

Signalling Peptides

SacB: MNIKKFAKQATVLTFTTALLAGGATQAFAKET

LipA: MKFVKRRIIALVTILMLSVTSLFALQPSAKAAEH

Epr: MKNMSCKLVVSVTLFFSFLTIGPLAHAQNS

References

1. Elastin Polypeptide pmid=11738083

1. Elastin Polypeptide pmid=11911775

2. Scaffold and EAK16-II pmid=16061392

3. Secretion in B.subtilis pmid=16997527

4. of Protein Secretion in B.subtilis pmid=10974125