Recombinant Surfactants Derived From the Naturally Occurring Protein Oleosin

Surfactants are molecules of key importance in the food, chemical and pharmaceutical industries due to their ability to stabilize interfaces and self-assemble in solution. Commercial surfactants are typically chemically synthesized making the material extremely difficult to functionalize for biological applications. Therefore there is a need to develop bioactive surfactants. A powerful alternative to chemical synthesis is the expression of recombinant protein surfactants through molecular biology. These proteins would be monodisperse and have the precise sequence dictated by the cognate gene. Recombinant protein production would permit the direct incorporation of specific motifs that mediate protein recognition. We have chosen the naturally occurring plant protein oleosin as a candidate. Oleosins are a family of plant proteins whose biological role is to stabilize oil bodies. They have two hydrophilic arms and a central hydrophobic domain. In this thesis, we have designed many variants of oleosin to self-assemble and/or stabilize interfaces. First we created a family of truncation mutants that assemble into sheets, fibers, or vesicles depending on the geometry of the protein and the solution chemistry. We further truncated this family to create protein variants that assembled into spherical micelles. All protein variants were confirmed using mass spectroscopy and the secondary structure was analyzed through circular dichroism. Protein aggregate size and shape were analyzed through light and X-ray scattering and directly visualized through cryogenic transmission electron microscopy. Oleosin variants were employed to stabilize interfaces in two applications. The first was the stabilization of microbubbles generated in a microfluidic device for ultrasound contrast and therapy. Bubbles were extremely stable over time, easily functionalized with an eGFP-oleosin fusion, and were echogenic. We further applied our variants to stabilize an oil-in-water emulsion for the creation of iron oxide nanoclusters as contrast agents for magnetic resonance imaging. Particles size was measured using light scattering and directly measured using electron microscopy. Oleosin stabilized iron oxide clusters were non-toxic and simple targeting to Her2/neu+ cells was achieved by blending a Her2/neu affibody-Oleosin fusion protein into the shell. These results set the foundation to further engineer oleosin for self-assembly and interface stabilization and functionalization