Proteins hold increasing promise for the development of new classes of analytical and therapeutic agents, but the cost of their enrichment and purification can be prohibitively high. Thus, there is a great need to develop improved processes for the purification of pharmacologically active proteins and peptides. Nature has already solved this kind of protein enrichment problem with the nuclear pore complex (NPC), the macromolecular complex that efficiently fractionates proteins between the cell nucleus and cytoplasm. Inspired by nature's solution, we have designed an artificial device that can sort nanoscale objects such as protein molecules and protein complexes and that can faithfully reproduce key features of trafficking through the NPC, including transport-factor-mediated cargo import. However, its performance still falls short of the NPC. Our goal is now to gain sufficient understanding of the molecular scale engineering principles behind nuclear transport to allow us to design next generation nanosorters capable of purifying any protein that we desire. We will first employ a """"""""reverse engineering"""""""" approach, dissecting the architecture and operation of the NPC to determine which parameters and structures of the NPC are essential to its main transport functions, and how these central parameters have been tuned by evolution to make transport so selective and efficient in the crowded cellular environment. We will also develop methods to assess in detail the performance and mechanism of our current machines in comparison with the equivalent parameters of the NPC, to determine how they work on a molecular scale and discover why the NPC still performs better, in particular with respect to selectivity. We will then use the information garnered in these experiments to guide the design of new and improved versions of our machines, with the aim of increasing their selectivity and efficiency. Our ultimate goal is to create robust, fully synthetic, high-speed protein purification devices. We will investigate how the principles of transport by the NPC can be used to purify any protein, offering potential alternatives to chromatography, filtration and precipitation as protein purification techniques to the biotechnology industry. We will also test the use of our nanosorting devices as components of advanced, mass spectrometry-based, proteomic analytical instruments.

Public Health Relevance

Almost all the active products from the biotechnology industry are proteins and peptides, but their purification remains a costly challenge. However, nature has already devised a rapid and efficient way to purify proteins;nuclear pore complexes sort many hundreds of proteins per second between the nucleus and cytoplasm. By discovering how nuclear pore complexes sort proteins, we are learning how to build prototype nanosorting machines, which eventually should be able to purify many kinds of medically relevant proteins and peptides far more efficiently and at far lower cost than currently possible.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BST-J (02))
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Ainsztein, Alexandra M
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Rockefeller University
Other Domestic Higher Education
New York
United States
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Lord, Christopher L; Timney, Benjamin L; Rout, Michael P et al. (2015) Altering nuclear pore complex function impacts longevity and mitochondrial function in S. cerevisiae. J Cell Biol 208:729-44
Hough, Loren E; Dutta, Kaushik; Sparks, Samuel et al. (2015) The molecular mechanism of nuclear transport revealed by atomic-scale measurements. Elife 4:
Kahms, Martin; Hüve, Jana; Peters, Reiner (2015) 4Pi microscopy of the nuclear pore complex. Methods Mol Biol 1251:193-211
Jovanovic-Talisman, Tijana; Chait, Brian T; Rout, Michael P (2014) NPC mimics: probing the mechanism of nucleocytoplasmic transport. Methods Cell Biol 122:379-93
Sampathkumar, Parthasarathy; Kim, Seung Joong; Upla, Paula et al. (2013) Structure, dynamics, evolution, and function of a major scaffold component in the nuclear pore complex. Structure 21:560-71
Niepel, Mario; Molloy, Kelly R; Williams, Rosemary et al. (2013) The nuclear basket proteins Mlp1p and Mlp2p are part of a dynamic interactome including Esc1p and the proteasome. Mol Biol Cell 24:3920-38
Fernandez-Martinez, Javier; Rout, Michael P (2012) A jumbo problem: mapping the structure and functions of the nuclear pore complex. Curr Opin Cell Biol 24:92-9
Fernandez-Martinez, Javier; Phillips, Jeremy; Sekedat, Matthew D et al. (2012) Structure-function mapping of a heptameric module in the nuclear pore complex. J Cell Biol 196:419-34
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Aitchison, John D; Rout, Michael P (2012) The yeast nuclear pore complex and transport through it. Genetics 190:855-83

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