Recombinant resilin

Resilin has a remarkably high fatigue lifetime (probably >500 million cycles) and our aim is to reproduce this desirable mechanical property in synthetic materials derived from our studies of resilin structure and function. We believe that recombinant resilin-like materials may be used, in the future, in the medical device field as components of prosthetic implants, including spinal disks and synthetic arteries. Spinal disks, for example, must survive for at least 100 million cycles of contraction and relaxation [30]. 

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

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]. 

Recombinant resilin production was induced, with the nonmetabolizable lactose analogue IPTG, in the Escherichia coli bacterial strain BL21(DE3)/pLysS. Cells were collected by centrifugation (10,000 g, 20 min at 4°C) and the cell pellet frozen at 80°C. 

From the DNA sequence of the resilin recombinant clone, the amino acid sequence of recombinant resilin can be deduced as... 

The calculated value for the Hisg-tagged protein is 28,492 Da. N-terminal amino acid sequence analysis (Procise) was carried out on purified recombinant resilin. The following sequence (at 120 pmol yield) was obtained for the first 12 amino acid residues MHHHHHHPEPPV, as expected from DNA sequence analysis. 

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. 

For recombinant resilin, we found that data from the CD spectra (Figure 9.4), x-ray diffraction pattern (Figure 9.5), and NMR were consistent with resilin being an amorphous random network... 

FIGURE 9.4 Circular dichroistn (CD) spectrum of dilute solution (0.24 mg/mL) of recombinant resilin in phosphate-buffered saline (PBS). (From Whelan, A.J. and Robinson, A.J., unpublished data). 

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 9.6 DSC of (a) recombinant resilin in water showing no enthalpic events, (b) bovine serum albumin in phosphate-buffered saline (PBS) showing denaturing occurring at 62°C, and (c) wool fiber in water showing denaturing of the a-helix at 145°C (Endotherm up). 

Structured proteins have also been investigated by thermal analysis [40,41], denaturing resulting in an endotherm which is readily detected by differential scanning calorimetry (DSC). DSC of recombinant resilin in the swollen state showed no transitions over a wide temperature range (25°C-140°C), further evidence of the absence of any strucmre. This is in contrast to the strucmred proteins wool and bovine serum albumin, which show denamration endotherms at 145°C and 62°C, respectively (Figure 9.6). 

In spite of the overwhelming evidence suggesting that recombinant resilin is amorphous, there are some results that suggest that a level of defined stmcmre cannot be completely ruled out. In particular, the fact that the protein solution coacervates when cooled (Figure 9.7) suggests that there is a degree of self-association between protein molecules. 

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]. 

FIGURE 9.7 Coacervated purified recombinant resilin. The lower phase of the aqueous solution contains about 300 mg/mL of purified recombinant resilin protein. The upper aqueous phase contains about 20 mg/mL protein. The protein solution separates at 4°C into a protein-rich lower phase. 

Tensile tests were carried out on recombinant resilin in PBS buffer on an Instron Tensile Tester (model 4500) at a rate of 5 mm/min and a temperature of 21°C. The swollen strip samples (7 X 1 mm) had a gauge length of 5 mm and were cycled initially up to a strain of about 200%. The... 

FIGURE 9.9 Small dumbbell-shaped sample of recombinant resilin prepared with embedded fine stainless steel mesh tabs (left) and being tested in phosphate-buffered saline (PBS) (right). 

FIGURE 9.10 In vitro cytotoxicity testing of individual components of recombinant resilin curing polymer system. The light gray areas represent green fluorescence, evidence of live cells, (a) Ammonium persulphate... 

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. 

No results have been acquired to date on the fatigue lifetime of recombinant resilin however, it is informative to consider the performance of natural resilin. 

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... 

A tendon from the wing region of the dragonfly was dissected and a cross-section with exposed resilin was mounted in PBS such that SPM force measurements could be made. The sample was shown to be 92% resilient [29]. When the technique was applied to recombinant resilin, the approach and retract curves were almost superimposed (Figure 9.18a). Analysis yielded a value... 

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). 

---