Hydrogels Based On The Specific Binding Between Proteins And Peptides And Their Applications

In recent years, self-assembly hydrogels of small molecules have attracted extensive research interest as a new type of biological materials. They have huge potential application values in various fields, such as three-dimensional (3D) cell culture and controlled drug delivery.During the formation of a molecular hydrogel, a small molecule (molecular hydrogelator) needs to selfassemble into a3D matrix of nanofibers, nanorods, or nanospheres that can hold water molecules within the cavities of the3D matrix through non covalent bond force (hydrogen bonding, wan der Waals force, electrostatic force, hydrophobic forces, π-π stacking interaction, cation π adsorption). To form the3D matrix, there should be strong or at least medium interactions between self-assembled nanostructures to maintain the self-assembly hydrogel stability and integrity. Otherwise, nanostructures with weak interactions between them will only form dispersions or solutions in the aqueous phase. Actually, there are many examples of this kind of self-assembled system that lack strong interactions between the self-assembled structures. Therefore, this type of solution/dispersion containing self-assembled nanostructures could change to a hydrogel if the interaction between the nanostructures could be enhanced.In this thesis, we rationally designed a fusion protein with four binding sites and used the protein-peptide interaction to enhance interactions between self-assembled nanofibers, thus leading to the formation of molecular hydrogels. This idea combined small molecular hydrogel of our group with protein hydrogel. It was the first time to propose such a novel stratege to form hydrogel. We designed a construct that could express the fusion protein ULD-TIP-1,and purified successfully the fusion protein ULD-TIP-1with four ligand-binding sites, In addition, we designed and synthesized a small molecule with a possible self-assembly ability (Nap-GFFYGGGWRESAI). Molecular hydrogels with three-dimensional network were formed by the special-interaction between a small molecule six peptide sequence WRESAI and the fusion protein ULD-TIP-1. The back of the experiment showed that it was important to form hydrogel with the three-dimensional structure. We used analytical ultracentrifuge to determined the molecular weight of the fusion protein ULD-TIP-1, and utilized ITC to detect the binding constant of the peptide-protein, the mechanics properties of the gel were studied by rheometer, and then the morphology of the compound solution and hydrogel were observed by AFM and cryo-TEM. Moreover, we also studied the phase transition between the hydrogel and solution. In conclusion, ULD-TIP-1can enhance the interactions of the self-assembly formation to form molecular hydrogel.Therefore, we used the specific protein-peptide interaction to enhance the interactions between self-assembled nanofibers, thus resulting in hydrogel formation. Furthermore, we can try to develop other kinds of protein-peptide hydrogels. Thus, we can explore the applications of the protein-peptide hydrogels in the field of biology.