Expression, cross-linking and characterization of recombinant chitin binding resilin

Abstract

Resilin is a polymeric rubber-like protein secreted by insects to specialized cuticle regions, in areas where high resilience and low stiffness are required. Resilin binds to the cuticle polysaccharide chitin via a chitin binding domain and is further polymerized through oxidation of the tyrosine residues resulting in the formation of dityrosine bridges and assembly of a high-performance protein-carbohydrate composite material. We describe for the first time a comprehensive study on the mechanical, structural and biochemical function of chitin binding recombinant Drosophila melanogaster resilin. Various resilin constructs were cloned including the full length gene enabling Ni-NTA purification, as well as heat and salt precipitation for rapid and efficient purification. The binding isotherms and constants (K d , B max ) of resilin to chitin via its chitin binding domain were determined and displayed high affinity to chitin, implying its important role in the assembly of the resilin-chitin composite. The structural and elastic properties were investigated using Fourier Transform Infrared Spectroscopy (FTIR), Circular Dichroism (CD) and AtomicForce Microscopy (AFM) with peroxidase crosslinked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine crosslinks. Further, the elastomeric properties found from the full length protein compared favorably with the shorter resilin generated previously from exon 1. The unusual elastomeric behavior of this protein suggests possible utility in biomaterials applications.

Biomaterials derived from silk–tropoelastin protein systems

“…The calculation of elasticity (resilience) has been discussed in detail for elastic resilin proteins in our previous studies [31]. The elasticity (resilience) calculated is defined as the energy recovered after removal of the stress, divided by the total energy of deformation.…”

Structural cuticular proteins from arthropods: Annotation, nomenclature, and sequence characteristics in the genomics era

“…They made an artificial protein with 16 copies of a consensus of the repeat in the first exon (GGRPDSYGAPGGGN), cross-linked it, and it too had elastic properties. Qin et al (2009) also carried out various physical measurements with the complete D. melanogaster protein and confirmed its elastomeric properties.…”

“…It is becoming common to use the term R&R Consensus for this expanded version and that practice is encouraged. There are three more published accounts that verify that this extended Consensus can confer chitin binding properties on a protein (Qin et al, 2009; Togawa et al, 2004; Togawa et al, 2007). Provocatively, Togawa et al (2004) also obtained chitin binding activity from shorter regions of the extended Consensus and other regions of the protein, but they used a low salt concentration in their binding buffer and had background binding from glutathione-S-transferase alone.…”

“…Dmelresilin has been demonstrated to bind chitin via its R&R Consensus region (Qin et al, 2009) and the authors speculate that a combination of bound and unbound resilin proteins contribute to the elastic properties. Alternatively, it may be that the two forms of resilin are used in different structures, for Bailey and Weis-Fogh (1961) report that the main-wing hinge of locusts had a “pad of pure rubber-like protein” in addition to a lamellar component with both chitin and resilin.…”

Clues for Biomimetics From Natural Composite Materials

“…Resilin is a rubber-like protein that displays stretching ability of up to 100% and is accompanied with a very high fatigue lifetime and low modulus of elasticity. Resilin is found especially in areas where high resilience and low stiffness are required, resulting in tough and durable structures [45,46]. Resilin binds to the cuticle chitin via the conserved cuticular ChBD type R&R and is further polymerized through oxidation of tyrosine residues resulting in the formation of dityrosine bridges leading to the assembly of high-performance protein–polysaccharide composites.…”