Cross-linked recombinant resilin

PRODUCTION OF CROSS-LINKED RECOMBINANT RESILIN 9.2.1 Synthesis... 

FIGURE 9.3 Cross-linked recombinant resilin. (a) Structure of the dityrosine adduct, (b) A cross-linked moulded rod. (c) HPLC analysis of acid-hydrolyzed uncross-Unked recombinant resilin and cross-linked recombinant resilin. (From Elvin, C.M., Carr, A.G., Huson, M.G., Maxwell, J.M., Pearson, R.D., Vuocolo 1, T., Liyon, N.E., Wong, D.C.C., Merritt, D.J., and Dixon, N.E., Nature, 437, 999, 2005.)... 

Amino acid analysis, by reverse-phase HPLC, of acid-hydrolyzed uncross-linked recombinant resilin and cross-linked recombinant resilin clearly shows the presence of dityrosine in the cross-linked sample (Figure 9.3c). Further evidence of the presence of dityrosine was obtained by UV irradiation (Xmax,ex 315 nm Xmax,em 409 nm). Dityrosine endows natural resilin with pH-dependent blue fluorescence [38] on UV irradiation. The cross-linked recombinant resilin material was similarly fluorescent, strongly suggesting dityrosine cross-links. 

FIGURE 9.5 X-ray diffraction pattern derived from a solid piece of cross-linked recombinant resilin. The lack of any sharp rings or features indicates that there is no distinct ordering in the sample. Some water scatter at about 3.5 A and the highest scattering around 6 A is observed. (From Pilling, P. and Varghese, J., unpublished data). 

FIGURE 9.18 Elastic properties of cross-linked recombinant resilin. (a) A single force-extension curve recorded for a sample of cross-linked recombinant resilin (approach curve solid, retract curve dotted). 

All elastic proteins contain distinct domains, of which at least one is made up of elastic repeat sequences, and they all contain cross-links between residues in either the nonelastic or elastic domains [9]. Previously, the Drosophila CGI5920 gene was tentatively identified as one encoding a resUin-like protein [31]. To prepare recombinant resilin, we chose to express the first exon of the Drosophila CG15920 gene [29], which encodes an N-terminal domain in the native protein comprising 17 copies of the putative elastic repeat motif GGRPSDSYGAPGGGN [31]. 

Other clues to the self-association of recombinant resilin in solution, and thus a degree of defined stmcture, include the propensity of the monomer proteins to covalently cross-link very rapidly through dityrosine side chains using a mthenium-based photochemical method [29]. Proteins which do not naturally self-associate do not form biomaterials when exposed to the Ru(ll)-based photochemical procedure (Elvin, C.E. and Brownlee, A.G., personal communication). Furthermore, Kodadek and colleagues showed that only intimately associated proteins are cross-linked via this zero-A photochemistry procedure [45]. 

In summary, none of the individual components necessary for recombinant resilin curing were cytotoxic, and there were no leachables from the cured resilin that caused cell death. The cured resilin polymer was not a good surface for cell adhesion, but cells can survive and proliferate in the resilin on a gelatine bead. The curing of recombinant resilin in the presence of cells on beads has no effect on the cells ability to migrate and proliferate with new tissue formation. The resilin is seen to degrade with time, but it is believed that this could be controlled by the type and extent of cross-linking. 

FIGURE 9.11 Relative mass change as a result of water absorption and loss in a solid piece of recombinant resilin prepared from a 20% protein solution in phosphate-buffered sahne (PBS), cross-linked using peroxidase (see [29] supplementary material). The fuUy swollen sample is designated 100. (Data courtesy of Shekibi, Y., Naim, K., Bastow, T.J., and HiU, A.J., The states of water in a protein based hydrogel. Internal CSIRO... 

In addition to conformational studies of RLPs, multiple groups have studied the mechanical properties of cross-linked RLPs. As mentioned above, Elvin et al. successfully expressed RLPs in E. coU in soluble form in 2005. This recombinant RLP was cross-linked via Ru(II)-mediated photochemical protocols to establish the formation of dityrosine cross-links in order to produce a cross-linked polymeric solid material at a concentration greater than lOOmgml". Tliis photo-cross-linking method is rapid, quantitative, and controllable, and its successful application suggests opportunities for multiple modes of cross-linking in engineering resilin-based materials. 

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