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Amino Acid Composition of Resilin

Amino Acid Constituents

Amino acid composition in Resilin was analyzed in 1961 by Bailey and Torkel Weis-Fogh when they observed samples of prealar arm and wing hinge ligaments of locusts . They summarize their data in Figure 1, below .

Fig. 1: Amino Acid Composition of Resilin

Figure 1 indicates that Resilin lacks Methionine, Hydroxyproline, and Cysteine constituents in its amino acid composition. Based on the structures of each amino acid in Figure 1, they were classified as being charged, hydrophilic, or hydrophobic. Based on the figure, It can be seen that Resilin has mostly charged and hydrophobic side chains.

Tyrosine Residues in Resilin

Andersen, in 1996, discovered that the tyrosine residues are involved in chemically covalent cross-links in many forms such as dityrosine, trityrosine, and tetratyrosine . Primarily, in Resilin, tyrosine and dityrosine served as the chemical cross-links, in which R groups of Tyrosine and Dityrosine add to the backbone of the growing peptide chain . Andersen came to this conclusion based on a study involving these two compounds in which he was able to rule out other forms of cross linking such as disulfide bridges, ester groups, and amide bonds . Though the mechanism of cross-linking of Tyrosine is understood that occurs through radical initiation, the cross linking of Resilin still remains a mystery. Cross linking of Resilin occurs very quickly and this is possibly a result of temperature. At increasing temperature, the rate of cross linking of the residues increases and leads to a highly cross-linked Resilin network .

In Figure 1, it can be seen that proline and glycine has a relatively high presence in the amino acid composition of Resilin. The presence of glycine and proline in the composition of Resilin contributes greatly to the elasticity of Resilin . Resilin, however, has an absence of an alpha-helix leading to a randomly coiled structure and a disordered structure . This is primarily due to the significantly high proline content in Resilin. Proline is a bulky amino acid that has the ability to cause a kink the peptide chain and due to the sterically hindered side chains, it is not able to fit in the alpha-helices. However, the segments of Resilin are able to take on secondary structure forms at different conditions.

Recombinant Resilin

Studies Involving Recombinant Resilin

With the study of Resilin stems the motivation for researchers to begin synthesizing recombinant Resilin type materials for various material applications. Recombinant Resilin was first studied in 2005 when it was expressed in Escherichia coli from the first exon of the Drosophila Melanogaster’s CG15920 gene . During its study, pure Resilin was synthesized into 20% protein-mass hydrogel and was cross-linked with ruthenium-catalyzed tyrosine in the presence of ultraviolet light .  This reaction yielded the product, recombinant Resilin (rec1-Resilin) .

Fluorescence of Recombinant Resilin

One unique property of rec1-Resilin is its ability to be identified due to autofluorescence. Fluorescence for Resilin stems primarily from dityrosine, which are the result of crosslinks of tyrosine residues. When ultraviolet light irradiates a sample of rec1-Resilin at 315 nm to 409 nm emissions, the rec1-Resilin begins to show blue fluorescence . An example of the blue fluorescence exhibited by the dityrosine residues in Resilin is shown in Figure 2, below. 

Property of Resilience

Another unique property of Resilin is its high resilience. Recombinant Resilin demonstrated excellent mechanical properties similar to that of pure Resilin. Elvin et al aimed to compare the resilience of rec1-Resilin to other rubbers, a scanning probe microscope of used. This study compared the resilience of rec1-Resilin to two different types of rubber: chlorobutyl rubber and polybutadiene rubber, both rubbers with high resilience properties . This study concluded that rec1-Resilin was 92% resilient compared to chlorobutyl rubber at 56% and polybutadiene rubber at 80%, respectively . With such high mechanical resilience, the properties of rec1-Resilin can be applied to other clinical applications within the field of Materials Engineering and Medicine. This study on recombinant Resilin has led to several years of research on the use of Resilin like proteins for several biomedical applications that retains the mechanical properties of Resilin.  The ongoing results of the studies involving recombinant Resilin may lead to further research in which other unexplored mechanical properties and chemical structure of Resilin may be investigated.

Clinical Applications of Resilin

Resilin-Based Proteins for Replacement of Heart Valves

Resilin has a variety of uses within the field of biomedical engineering and medicine. Its unique properties has offered researchers many alternative replacements to existing materials used within scientific research. One potential use of Resilin is for the replacement of heart valves. With the rise in emerging diseases and disorders, there has been a significant interest within the scientific community to use protein-based tissue engineering methods to target and treat many diseases and disorders that affect the population. It was previously discussed in a section above that, Resilin is primarily regarded as a hydrogel, a network of cross-linked polymer chains that are capable of swelling when a hydrophilic solvent such as water is used as a medium. Hydrogels have been studied extensively and have been used as carrier systems for appropriate drug delivery and for targeted tissue engineering applications in many areas of the body. In a study done at the School of Chemical Engineering and Weldon School of Biomedical Engineering at Purdue University, the use of Resilin-based recombinant proteins were explored since the mechanical properties of these proteins could be modified as and when needed .  Many cross-linking methods have been explored so far such as photo-initiated crosslinking methods or Click Chemistry with a poly (ethylene glycol) (PEG) base system. In this study, however, Kim et al used a mild cross-linking chemistry recombinant Resilin hydrogels with the aim of expanding the utility of Resilin in Tissue Engineering applications . The authors of this study utilized transglutaminase, an enzyme found in many tissues, as the chemical cross-linker. Transglutaminase has been widely used in blood coagulation or wound healing applications . The study aimed to utilize Resilin-based hydrogels cross-linked with transglutaminase for cell viability, cell differentiation, and cell attachment studies . The authors aimed to create mixtures of stiff two-dimensional hydrogel matrices by varying the protein concentration during production in order to understand how the cell response is affected by matrix stiffness . The authors analyzed the swelling ratios and contact angle through several tests in which the synthesized hydrogels were placed in a solution that contained phosphate-buffered solution (PBS) for a period of time . The result of these tests indicated that as the stiffness or as the protein concentration increased, the swelling ratio and contact angle of the hydrogel decreased . Through dead/live assays and cell proliferation assays, the authors of this study were able conclude that stiffer hydrogel matrices were better at promoting cell adhesion and cell spreading compared to softer hydrogel matrices and other cross-linkers . The results obtained from this study can be used mimic endothelial cell attachment in blood vessels ^[8]. In future studies, if cell attachment is possible, then it can be used for targeted therapy for cardiovascular applications such as for regeneration of damaged heart valves.

Resilin-Based Proteins for Regeneration of Load-Bearing Cartilage

Based on the understanding of the mechanical properties of Resilin, it is understood that Resilin demonstrates 92% resilience meaning that it is highly elastic protein able to store energy and release it at different time increments as it is able to return to its original shape. This unique property of Resilin has been applied to cartilage in the human body with aims of regeneration. The wear and tear of cartilage can lead to many debilitating diseases that affect the human body. One such example is osteoarthritis, a debilitating disease that affects millions of people, including the geriatric population, around the world. This degenerative disease occurs when the cartilage surrounding the body tears or is worn out as a result of excessive or repetitive strain. Researchers currently are using various regeneration methods using stem cell therapy as ways to repair the cartilage. One study, however, by Li et al aims to apply the mechanical properties of Resilin to repairing cartilage. In this study, synthesized recombinant Resilin-like hydrogels that are comparable to the mechanical properties of natural Resilin . The authors then added RGD cell-binding domains to the Resilin-based proteins and cross-linked using Tris (hydroxymethyl phosphine) and synthesized mechanically stable hydrogels capable of human mesenchymal stem cell attachment . The results of this study suggested that the relatively stable Resilin-like hydrogels can be used for effective regeneration of cartilage at an injured site with the desired mechanical properties . If targeted studies done through clinical trials are successful, then it be could be applied as a form of treatment for millions of people around the world and as a result improve the quality of life they see.

Resilin-Based Proteins for Vocal Cord Treatment

The unique mechanical properties of Resilin have been explored in the treatment for vocal cord injuries as well. Mechanical and environmental stress on the vocal cords or a preexisting morbid conditions can affect the pliability of lamina propria of vocal folds . Lamina propria is the control house for the production of sound and affects many people around the world . Currently, surgical techniques are invasive and are used to treat the symptoms associated with vocal cord injuries but it often scars the surrounding tissue . In order to minimize the scarring and to make the surgical treatment minimally invasive, researchers are using tissue regeneration methods to treat these injuries. In a study by Teller et al, they utilized the mechanical properties of purified Resilin-like hydrogels and cross-linked the hydrogels with [tris (hydroxymethyl) phosphine] propionic acid using a Mannich-type reaction .  The result was the formation of stable hydrogels that retained the excellent properties of pure Resilin such as its resilience and stability . The results of the study indicated that Resilin-based hydrogels are relatively stable and yield high mechanical properties and can be utilized for tissue regeneration applications to vocal cords.