말미잘 유래 반복서열 단백질 기반 바이오소재 연구

Abstract

Protein-based biomaterials have been developed for various biomedical applications by mimicking mechanical, physical, and biological properties of natural extracellular microenvironments. Although protein-based biomaterials provide superior biocompatibility and biofunctionality, unfortunately, they have low mechanical properties compared with real tissues. To promote enhanced mechanical properties, repeat proteins have been regarded as the best candidates among diverse proteins for biomaterial component, because most repeat proteins possess welldefined secondary structures which affect mechanical characteristics and structural stability of material. In this thesis, new repeat proteins from sea anemone were discovered. Through genetic design, production, and fabrication, the mechanical, physical, and biological characteristics were analyzed. Based on the results, sea anemone-derived repeat proteins would be successfully applied in diverse tissue engineering applications.

First, a hypothetical repeat protein sequence which has sequence similarity with Bombyx mori fibrous proteins, collagen, and flagelliform spider silk was found. To find location of the target protein in sea anemone's tissue, 15-mer partial amino-acid sequence (PGQGPGNTGYPGQGP) of protein and its antibody were synthesized and used in immunohistochemical analysis. The repeat protein was dominantly detected in the tentacles as well as its skin. The repetitive DNA sequence of protein (named aneroin) was redesigned to avoid deletion or premature termination problems. The recombinant aneroin with molecular weight of ~32 kDa, ~64 kDa, and ~128 kDa (designated aneroin-30K, 60K, and 128K, respectively) were constructed. Due to low expression of aneroin-128K, only aneroin-30K and aneroin-60K were employed for further experiments. Affinity-based purification and heat/acid-based purification were applied for aneroin purification. Heat/acid-based purification method was much simple and easy, and its purity was confirmed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and amino acid composition analyses. Through wet- and electrospinning, aneroin was spun into microfiber and nanofiber sheet, respectively. The mechanical properties of aneroin microfibers were similar to those of wet-spun fibers from recombinant spider silk proteins, with molecular weights of ~100 kDa of the major ampullate spidroin I monomer and natural mammalian tendon collagen. This result demonstrated the possibility of repeat protein-based fiber as a tissue engineering scaffold based on its reliable productivity and mechanical property.

Second, the specific application field of aneroin was investigated. The field where mechanical property is main limitation was hydrogel-based protein scaffold aiming artificial skin. Therefore, using ~5% of tyrosine residues in aneroin, aneroin was successfully photo-crosslinked and transformed into 3D swellable hydrogel. Additionally, thiol-disulfide exchange reaction, with reduced and oxidized glutathione (GSH/GSSG), was applied to promote disulfide crosslinking for more strong mechanical features. Without disulfide crosslinking promotion, the aneroin hydrogel showed ~80 kPa of tensile stress and ~190 kPa of elastic modulus. Whereas, in the presence of disulfide crosslinking promotion, the hydrogel demonstrated ~3 times greater stress (~300 kPa) and elastic modulus (~550 kPa). It was higher mechanical stress compared to alginate or gelatin which are widely applied materials in artificial organ, and similar value to aorta (~300-800 kPa). In terms of mechanical stress, those values were insufficient for replacing real skin (~1-20 MPa). However, in biological point of view, elastic modulus is much important than stress because surface elastic modulus greatly affects cellular behaviors. For aneroin hydrogel, it exhibited similar elastic modulus of silk hydrogel (4-400 kPa) or skin (10-100 kPa), and higher elastic modulus than those of collagen (~1.8 kPa) and fibrin gel (3-80 kPa). The mechanical property of aneroin was expected to provide proper elastic modulus for fibroblast or epidermal cells which are largely distributed in skin, and it was experimently proved by direct injection of HaCaT and NIH/3T3 cells. These results demonstrated that the aneroin-based hydrogel would be the appropriate scaffold to cultivate skin cells based on skin-like elastic modulus and strain (extensibility).

Additionally, another repeat protein (anegen) from sea anemone's skin was discovered. Anegen has ~30% of similarity with spore coat protein, proline-rich cell wall protein, and mussel's foot protein, but most protein sequences matched with anegen were uncharacterized proteins. The anegen showed highly biased amino acid composition (six amino acid types occupied about 85% of total amino acids) with strictly arranged decamer repeats. Through CD analysis, it was confirmed that representative decamer repeat of anegen possesses polyproline II conformation, which is the prevalent conformation found in elastomeric proteins such as elastin, titin, abductin, lampirin (ECM protein), and collagen. The recombinant anegen (~40 kDa) was extracted with formic acid, and the purity was confirmed via reverse-phase highperformance liquid chromatography (HPLC) and amino acid composition analyses. Because polyproline II can be converted from b-strand to b-turn reversibly using its hinge structure, we assumed that the extended repeats in recombinant anegen would involve in conformational flexibility and elasticity of protein. Therefore, we specified application field of anegen into cartilage replacement because cartilage should endure cyclic mechanical stresses everyday. Based on high tyrosine contents (~10%), dityrosine crosslinkage of anegen within various crosslinking density was induced. Through cross-linking density variation, tensile stress was increased about 5 times, and that of elastic modulus was about 10 times. The best mechanical stress was ~2.3 MPa with ~5.3 MPa of elastic modulus and 76% of strain. Anegen hydrogel presented higher ultimate stress than other tissue engineered cartilages (less than 1 MPa) as well as higher or comparable elastic modulus (1-5 MPa). In addition, there was no severe material deformation under 20 times of cyclic compression. Thus, water-haboring but not swellable anegen hydrogel exhibited stable tensile and compressive mechanical characteristics as a load-bearing hydrogel, especially for the potential application of hydrogel-based cartilage substitute.

Collectively, we developed protein-based scaffolds with two novel repeat proteins (aneroin and anegen) which were assumed to be involved in shrinkage behavior of sea anemone. Through mechanical, physical, and biological property analyses, we revealed those unique haracteristics and specified application area of those repeat proteins. Aneroin and anegen exceeded properties of some protein- or natural-based biomaterials which were widely employed in tissue engineering applications. Although it cannot be comparable with real skin or cartilage yet, it would be extensively developed by optimizing several variables affecting mechanical or biological properties. Given that different protein composition and cell distribution in our body, new repeat unit would be advantageous not only for detecting unforeseen properties but also for overcoming previous limitations of protein-based biomaterials.