Functionalised Silk Fibres
Final rept. 1 Aug 2009-30 Jul 2012
COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANIZATION CANBERRA (AUSTRALIA) DIV OF ECOSYSTEM SCIENCES
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The aim of this project was to understand the structure-properties-performance relationship of honeybee silks and to use this information to guide design of second-generation honeybee silk materials. We demonstrated a single recombinant silk protein could adopt the native silk molecular structure and be fabricated into materials with equivalent mechanical properties to that generated from recombinant proteins produced from the four silk genes found in honeybees. We selected amino acids for specific alanine to cysteine mutations and then used site-directed mutagenesis followed by recombinant expression to make these mutants in E. coli. We demonstrated that mutation did not affect our ability to produce the protein at high levels, purify them to generate concentrated silk protein solutions, or our ability to fold them into the native molecular structure. These results indicate that the protein is not under strong sequence constraint and is ideally suited for development of second-generation protein materials. Another major part of this project investigated the molecular structure of native honeybee silk in more detail. We demonstrated that native silk contains beta-sheet structures as well as the previously described coil molecular structure. The presence of beta-sheets is insufficient to explain the mechanical performance of the material, so we examined the possibility of cross-linking between protein chains in native silk. Amino acid analysis of the material demonstrated a deficiency in lysine residues in comparison to that predicted from protein sequences. Analysis of native proteins within the silk glands indicated the presence of covalent cross-links before silk fibre fabrication. We refined the coiled coil molecular structural model of honeybee silk to include low levels of both beta-sheet and covalent cross-links. We developed a range of bio-mimetic silk materials and demonstrated the materials had similar structure and performance to native silks.
- Genetic Engineering and Molecular Biology