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Research Projects

Virtual Gating Machines for Protein Purification

Most commercial large-scale production of proteins uses column chromatography and synthetic membranes to fractionate and concentrate proteins. Though the requisite multiple recovery steps increase purity, yields drop quickly and expenses rise. Improving the performance of these processes is therefore a high priority. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular machine that efficiently segregates proteins between the nucleus and cytoplasm of all eukaryotic cells. Since we now have an understanding of the mechanism by which the NPC transports proteins, our goal is to mimic the molecular machinery of the NPC, with its exquisite selectivity and high throughput, in a robust synthetic platform. Our approach is one of reverse engineering, in which we will dissect the nuclear transport system to elucidate its key elements in order to duplicate them. We are using the yeast NPC, the best understood system, as our starting point. By disrupting transport in vivo and observing the resulting kinetic changes, we are identifying those NPC components that are essential for transport and learn how those components function. By also determining the transport properties of key individual NPC components, we aim to define the "minimal machine" that we will then seek to mimic.

Diagram illustrating two possible virtual gating membrane configurations.
Diagram illustrating two possible virtual gating membrane configurations.
(Left), FG Nups (green) deposited locally around nanopore on top surface of membrane (blue). (Right), FG Nups deposited uniformly across top surface of membrane. Bacterial lysate containing carrier tagged cargo (orange) among non-specific lysate proteins (gray) is placed above the membrane, and the cargo is preferentially segregated to below the membrane.