Directed Self-Assembly of Heterologously Expressed Hagfish EsTKα and EsTKγ for Functional Hydrogel

Abstract

Hagfish slime proteins have long been considered useful due to their potential applications in novel green, environmental, and functional bionic materials. The two main component proteins in the slime thread of hagfish, (opt)EsTKα and (opt)EsTKγ, were used as raw materials. However, the methods available to assemble these two proteins are time- and labor-intensive. The conditions affecting protein self-assembly, such as the pH of the assembly buffer, protein concentration, and the protein addition ratio, were the subject of the present research. Through a series of tests, the self-assembly results of a variety of assembly conditions were explored. Finally, a simplified protein self-assembly method was identified that allows for simple, direct assembly of the two proteins directly. This method does not require protein purification. Under the optimal assembly conditions obtained by exploration, a new gel material was synthesized from the hagfish protein through self-assembly of the (opt)EsTKα and (opt)EsTKγ. This assembly method has the benefits of being a simple, time-saving, and efficient. The self-assembled protein gel products were verified by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and contained (opt)EsTKα and (opt)EsTKγ proteins. Scanning electron microscopy (SEM) was used to investigate the self-assembled protein gel after freeze-drying, and it was observed that the self-assembled protein formed a dense, three-dimensional porous network structure, meaning that it had good water retention. Evaluation of the gel with atomic force microscopy (AFM) indicated that the surface of the protein fiber skeleton show the network-like structure and relatively smooth. Characterization by circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR) demonstrated that the two proteins were successfully assembled, and that the assembled protein had a secondary structure dominated by α-helices. The rheological properties of the self-assembled products were tested to confirm that they were indeed hydrogel property.