388378 Artificially Engineered Protein Gels Derived from Nucleoporins

Thursday, November 20, 2014: 1:14 PM
International 7 (Marriott Marquis Atlanta)
Minkyu Kim1, Wesley Chen2, Jeon Woong Kang3, Matthew J. Glassman1, Katharina Ribbeck2 and Bradley D. Olsen1, (1)Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA, (2)Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, (3)Laser Biomedical Research Center, G. R. Harrison Spectroscopy Laboratory, Massachusetts Institute of Technology, Cambridge, MA

The nuclear pore, a tiny channel 45 nm in diameter, is filled with a polypeptide-based hydrogel capable of selectively filtering < 0.1% of all proteins in a eukaryotic cell while translocating over 1,000 molecules per second.  The responsible polypeptides are nucleoporins, commonly containing “FG” repeat sequences, which are known to associate with one another to construct physical gels and give rise to the hydrogel’s selective filtering property. Major obstacles in utilizing nucleoporin-based hydrogels for separation technology include low biosynthetic yield and long gel processing time, which typically takes several hours to days. 

In order to overcome these drawbacks, we developed artificially engineered protein building blocks that attempt to mimic the selective permeability of nuclear pore hydrogels, are produced in high yields, and possess reduced gelation times. A well-investigated nucleoporin, Nsp1, was used as a template sequence for its well-defined repeat domains. To mimic Nsp1’s transport properties, nucleoporin-like polypeptides (NLPs) were designed from consensus Nsp1 repeat sequences. To facilitate hydrogel formation, NLP sequences were flanked with coiled-coil domains which create three dimensional polymer networks.  Here, we demonstrate that artificially engineered biopolymer building blocks can be expressed in high yield and can construct a physically crosslinked hydrogel within a few seconds. Using a combination of selective permeability fluorescence assays, shear rheology, and Raman spectroscopy, the mechanism of artificially engineered NLP hydrogel function is studied.

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See more of this Session: Naturally-Derived and Sustainable Biomaterials
See more of this Group/Topical: Materials Engineering and Sciences Division