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 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, circular dichroism, and atomic force microscopy with peroxidase cross-linked solid resilin materials. Generally, little structural organization was found by these biophysical methods, suggesting structural order was not induced by the dityrosine cross-links. 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.

Molecular and functional characterisation of resilin across three insect orders

“…Although D. melanogaster isoform A transcripts have been expressed successfully (Qin et al, 2009), these longer transcripts do not express as well as the shorter transcripts in our laboratory (unpublished data). Furthermore, Qin et al (2009) observed that the heat and salt purification method that we employ in the current paper abolished chitin-binding ability of the D. melanogaster isoform A recombinant proteins. As our goal was to generate recombinant proteins for biomaterial fabrication, we were most interested in the isoforms likely to give us greatest expression.…”

“…Previous studies of the secondary structure of resilin-like proteins suggest that they are unstructured with a high degree of disorder dominated by random coil configurations (Lyons et al, 2009;Nairn et al, 2008;Qin et al, 2009). To test that Cf-resB and Hi-resB have similar structural characteristics, we gathered secondary structure information using CD spectra and prediction of intrinsically-unstructured regions of proteins based on estimated energy content (Dosztanyi et al, 2005) (http://iupred.enzim.hu/).…”

A buckling region in locust hindlegs contains resilin and absorbs energy when jumping or kicking goes wrong

“…In all preparations, fluorescence was dependent on pH, a second characteristic signature of resilin (Andersen, 1963;Neff et al, 2001). Resilin is likely to be cross-linked with other proteins in the cuticle (Qin et al, 2009).…”

Expression of the rubber‐like protein, resilin, in developing and functional insect cuticle determined using a Drosophila anti‐rec 1 resilin antibody

“…Resilin-bearing cuticle also acts to counteract movement caused by muscles, such as in the opening mechanism of the spiracle in locusts (Miller, 1960), the sound-producing tymbals in cicadas (Young and Bennet-Clark, 1995), and the joints connecting segments in the legs of the cockroach, Periplaneta americana (Neff et al, 2000). The development of biomimetic resilin-like compounds (Elvin et al, 2005;Qin et al, 2009) and their possible applications (Kopecek, 2007) means that the methods by which insects utilise resilin will come under more intense scrutiny. Most studies that identify resilin-rich regions rely on the optical and chemical properties of resilin, combined with an elasticity or energy storage function.…”