General applications

ELPs are protein polymers derived from human tropoelastin. One of their key features is that they exhibit a phase separation that is often reversible whereby samples remain soluble below Tt but form coacervates above Tt. They have many possible applications in purification, sensing, activation, and nano assembly. Furthermore, they are non-immunogenic, substrates for proteolytic biodegradation, and can be decorated with pharmacologically active peptides, proteins, and small molecules. Recombinant synthesis additionally allows precise control over ELP architecture and molecular weight, resulting in protein polymers with uniform physicochemical properties suited to the design of multifunctional biologics. As such, ELPs have been employed for various uses including as anti-cancer agents, ocular drug delivery vehicles, and protein trafficking modulators³.

Construct design

The construct consists of ELPs and FKBP12. In general, ELPs have hydrophilic and hydrophobic domains that exhibit reversible phase separation behavior that is temperature-dependent. They are made from a repeating VPGXG sequence, with X replaced by specific amino acids. This results in specific properties of the ELPs, especially related to the transition temperature Tt at which the ELPs will interact with each other on the hydrophobic sites². When the temperature is below Tt, the water molecules surrounding the hydrophobic parts will go into the bulk water phase which gains the solvent entropy. This makes it possible to form interactions with other ELP molecules³.

This construct has a hydrophilic region in the middle (A[120]) and a hydrophobic region on each side of it (I[60]). On the ends, FKBP12 domains are located for stronger interactions between the ELPs. FKBP12 can form a complex with Rapamycin and the FKBP-rapamycin binding (FRB) domain. This mechanism is normally used in the mTOR pathway inside cells, which regulates cellular proliferation, protein synthesis, differentiation and survival, and lipid metabolism. In the case of a hydrogel, the addition of rapamycin to a cell with these ELPs and ELPs with FRB at the ends can induce crosslinking between them to form a network (bron inobe en yandong liu). These stronger interactions make them useful in the formation of a hydrogel.

As soon as the hydrogel is made inside E.coli BL21 cells, the cells are prevented from dividing. However, the cells remain functional. So they can still be used to express therapeutic agents, like Interleukin 10 in the TU Eindhoven 2023 teams project.

Results

References