Biomimetic engineering of shock-absorbing materials inspired by marine snail egg capsule proteins

• Main Author: Fu, Tianpei
• Other Authors: Ali Miserez
• Format: Theses and Dissertations
• Language: English
• Published: 2015
• Subjects: DRNTU::Engineering::Materials
• Online Access: http://hdl.handle.net/10356/62333
• Institution: Nanyang Technological University

The egg capsule from the marine snail Busycotypus canaliculatus is a bioelastomer that possesses unique biomechanical properties. It is highly elastic and relatively stiff in comparison to both synthetic and natural elastomeric materials, and exhibits a remarkable capability to absorb mechanical energy during loading/unloading cycles. The elasticity is instantaneous and fully reversible, and the cycling loading can be repeated hundreds of times without any internal damage. These properties make the egg capsule an intriguing model for the design of biomimetic shock-absorbing materials that can be used in applications such as tissue engineering. The egg capsules are made of mostly proteinaceous fibers, which consist of α-helical coiled-coils. When the egg capsules are subjected to an external force, the α-helices undergo a transition into β-sheets, which is directly related to the elasticity of the capsular material. In stark contrast to common bioelastomers, such as elastin or resilin, the driving force for this process is not entropic but instead is mainly governed by changes in the internal energy associated with conformational changes of the peptidic backbone. Four egg capsule proteins (ECPs) have been identified from the nidamental gland of female snails and were recently sequenced. In this project, we aimed to set up a recombinant expression system to produce ECPs, use these proteins to study their self-assembly into coiled-coils filaments, and then engineer biomimetic materials mimicking the load-bearing characteristics of the egg capsule. We cloned the genes encoding the ECPs and expressed the recombinant proteins in E. coli. The proteins were exclusively expressed in the insoluble inclusion bodies of the bacteria. They were thus extracted with a high concentration of denaturants, and purified by strong cation exchange chromatography under denaturing conditions. They were subsequently refolded in aqueous solutions by a dialysis strategy. The secondary structures of the refolded proteins were analyzed by Circular Dichroism (CD) and Fourier Transform Infrared spectroscopy (FTIR). These studies revealed that ECPs assembled into heteromeric coiled-coils. High-resolution Atomic Force Microscopy (AFM) suggested the coiled-coils were hetero-dimers, similar to the coiled-coils in the intermediate filament (IF) proteins keratins. The thermostability of the refolded proteins was assessed by temperature-dependent CD and the coiled-coils were found be highly stable. The diameter of covalently fixed coiled-coils was determined by Transmission Electron Microscopy (TEM), further suggesting similarity with the self-assembly of keratins and other IFs. ECP coiled-coils were cross-linked by amine cross-linking reagents and the resulting products exhibited a mechanical response that approached that of the native egg capsule as determined by depth-sensing nanoindentation. Successful elucidation of the coiled-coils assembly mechanisms and initial production of mechanically robust materials represent a first key step towards the engineering of biomimetic shock-absorbing materials based on marine snail egg capsules.