Developing a novel multi-functional material

Discovery of a novel protein-based material Ultrabithorax (Ubx) is a Drosophila transcription factor that was discovered to form materials by our group in 2006. During protein purification it was noticed that protein elution drops hung from the column through an “invisible” thread (Fig 1A), which was actually Ubx protein fiber.

Material production Ubx was recombinantly produced in E.coli and purified via His-tag affinity purification. To induce material formation, purified Ubx protein solution was placed on a siliconized surface to form a drop. At the air-water interface, Ubx monomers self-assemble into Ubx film (Fig 1B), which can be spun into Ubx fibers via weak mechanical forces (Fig 1C).

Figure 1. A) Discovery of Ubx-based materials. B) Ubx film form spontaneously at the air-water interface. C) Production of Ubx fiber from Ubx film via pulling.

Producing various morphologies Depending on the production method, Ubx-based materials can adopt a number of morphologies. Films (Fig 2A) form via self-assembly at the air-water interface; fibers (Fig 2B) are spun from films (Fig 2B); bundles (Fig 2C) are fused together by fibers.

Figure 2. Multiple morphologies of Ubx-based materials: A) an Ubx fiber; B) an Ubx bundle composed of Ubx fibers fused together; C) Ubx film.

Incorporation of protein functions into materials An innovative feature of Ubx-based materials is the ability to incorporate another functional protein (e.g., GFP) into the material itself. I achieved this by fusing protein of interest, together with a linker sequence, to the N-terminus of Ubx. I designed the plasmid encoding this chimera and used E.coli for recombinant protein production (Fig 3A, 3B). The resultant chimeric protein produced material with fluorescent properties (Fig 3C).

Figure 3. Incorporating functional proteins into Ubx-based materials. A) Designing the plasmids encoding green and cherry fluorescent proteins fused to Ubx for recombinant production in E.coli. B) Purified fusion chimeras in side-by-side drops. C) Fluorescent materials can be produced from fusion chimeras.

Generation of functional patterns within materials The spatial arrangement of bioactive molecules (such as cadherin and ephrins) is key to the tissue formation but remains a challenge in the field of tissue engineering. Using fluorescent-Ubx fusion as probes, I achieved microscale control over the distribution of protein molecules within Ubx-based materials (Fig 4 A-C).

Figure 4. Patterning of functional Ubx chimera materials: A) Production of a striped fiber; B) Production of a faced fiber; C) Production of a twist.

Applications in development I am developing two applications based on these existing technologies:
• A tissue engineering scaffold that has built-in functional protein molecules
• A heme sensor that changes color upon detecting heme