Synthesis and properties of crosslinked recombinant pro-resilin

Abstract

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications.

Chemoenzymatic Synthesis of Polypeptides for Use as Functional and Structural Materials

“…Methyl, [57,58,63] ethyl [46,49,52,56,62] Proteinase I, [49] papain (4)(5)(6)(7)(8)(9)(10)(11)(12), [46,52,[56][57][58][59][60]62] bromelain (6.5-7.9), [46,59] α-chymotrypsin (8.2-9.4), [46,59,61] protease SG (8.5), [59] CPDY (6) [63] l-Isoleucine…”

“…Ethyl, [49] isopropyl [23,24] Proteinase I, [49] papain (5)(6)(7)(8)(9)(10)(11)(12)(13)(14), [60,67,68] α-chymotrypsin [23,24] l-Lysine…”

“…The most representative example is resilin, a structural protein that is found in insects, such as dragonflies and fleas. [9,10] Resilin comprises randomly coiled polypeptide chains that are cross-linked by multiple di-and trityrosine moieties and displays outstanding physical properties, such as extremely high elasticity and high fatigue lifetime. Another example can be found in melanins, a family of natural pigments produced by the polymerization of tyrosine-based molecules that protect living forms from photoinduced damages.…”

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.…”

“…While Drosophila pro-resilin was the first insect resilin gene to be completely sequenced, to date there has been no direct confirmation that resilin is indeed expressed as a cuticular protein in Drosophila as inferred from the chitin-binding domain (R&R Consensus) (Andersen, 2010). We use an antibody generated against exon 1 of recombinant Drosophila resilin (Elvin et al, 2005) to show that resilin is expressed in the developing larval epidermis of Drosophila and we identify sites of expression in the developing adult cuticle of late-stage pupae. We show that the antibody cross-reacts, binding to sites of suspected or known resilin expression in other insect species, and therefore has wide application in confirming the presence of resilin and allowing investigation of its spatial and temporal expression.…”