Resilin protein

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. 

Chapter 9 Recombinant Resilin—Protein-Based Elastomer.255... 

The reel-resilin protein has also seen application in other areas. The ability to control the organization of proteins to various surfaces is crucial in many biological... 

Exotic proteins Monellin Resilin Glue proteins... 

Some proteins display rather exotic functions that do not quite fit the previous classifications. Monellin, a protein found in an African plant, has a very sweet taste and is being considered as an artificial sweetener for human consumption. Resilin, a protein having exceptional elastic properties, is found in... 

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]. 

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. 

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 9.11 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... 

Weis-Fogh, T., Molecular interpretation of the elasticity of resilin, a rubber-Uke protein, J. Mol. Biol., 3, 520-531, 1961. 

Lyons, R.E., Lesieur, E., Kim, M., Wong, D., Brownlee, A., Pearson, R., and Elvin, C., The development of synthetic genes encoding repetitive resilin-like polypeptides Construct design, bacterial expression and rapid purification. Protein Eng. Design Select., 20(1), 25-32, 2007. 

Andersen, S.O., Covalent cross-links in a structural protein, resilin, Acta Physiol. Scand. Suppl., 263, 1-81, 1966. 

The amino acid composition of resilin was elucidated shortly after its first description and was shown to be unique among other structural proteins found in Nature a summary of the amino acid sequence is given in Table 1. 

Resilin and elastin, unlike other structural proteins, fulfill both definitions of an elastic material. Colloquially speaking, resilin and elastin are stretchy or flexible. They also fulfill the strict definition of an elastic material, i.e., the ability to deform in proportion to the magnitude of an applied stress without a loss of energy, and the recovery of the material to its original state when that stress is removed. Resilin and elastin are alone in the category of structural proteins (e.g., collagen, silk, etc.) in that they have the correct blend of physical properties that allow the proteins to fulfill both definitions of elasticity. Both proteins have high extensibility and combine that property with remarkable resilience [208]. 

Resilin and elastin have relatively high extensibility and resilience, but as compared to the collagen and the silks, the proteins sacrifice stiffness (elastic modulus) and strength (see Table 2). Collagen and dragUne sflk are much stiffer materials, but lack the extensibility that is characteristic of the rubber-like proteins. On the other hand, the mussel byssus fibers and the viscid silk have the extensibility of resilin and elastin, but lack the resilience [208]. 

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