1987, University of Virginia
School of Medicine
Office: 153 Robert F. Poole Agricultural Center Phone: (864) 656-3586
Research Focus Areas
Natural and synthetic fibrous molecules provide some of the most durable materials known. Much of the synthetic fiber industry has focused on the development and production of hydrocarbon-based materials including nylon, polyethylene and polypropylene. While many of these high-performance fibers exhibit desirable physical properties, their production requires the use of hazardous organic solvents as well as elevated temperatures and pressures. In contrast, there are a number of naturally-occurring fibrous materials that are spun at ambient temperature and pressure from an aqueous solution. Among these are the highly insoluble proteinaceous silk fibers produced by numerous species of insects and spiders. The best-studied silks are those of the common silkworm and the orb-weaving spiders. Spider dragline silk exhibits appreciable elasticity and strength resulting in an incredibly tough fiber. It is used by the spider not only to construct the outer frame and radii of the web but also as a hanging lifeline that allows the spider to evade and/or escape from predators. The core constituents of dragline silk are two fibrous proteins produced in the major ampullate gland that are called major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). These proteins consist of a large central repetitive domain flanked on both the N- and C-terminus by non-repetitive domains. Interestingly, while the repeat domains vary greatly among silks with different mechanical properties, the N-terminal and C-terminal domains found on mature spidroins are not only conserved between MaSp1 and MaSp2, but also among many silk types and spider species. This suggests that despite their relatively small size, they play an important and conserved role for the function of the silk. Since the one thing all silks must do is form a fiber, we hypothesize that the non-repetitive domains are instrumental in fiber assembly. It is our goal, and the focus of study in this lab, to try to understand the mechanism that leads from soluble protein to an insoluble fiber. The ideal situation would allow use of full-length, native fibrous proteins for direct assessment of structure-function relationships and assembly mechanisms. However, certain aspects of spider biology and behavior preclude their use as a source of material. First, spiders produce only miniscule amounts of spidroin proteins. Second, even if sufficient material was produced, their territorial and cannibalistic behavior precludes maintenance of spider colonies. As a result, we have used recombinant DNA technology to clone the non-repetitive sequences of native MaSp1 and MaSp2 from Nephila clavipes and are able to produce significant amounts of native or tailored fibrous protein domains and mini-silk proteins in vitro.
Using a variety of molecular techniques we are evaluating interactions among various protein domains to further our understanding of how these molecules perform in the natural protein polymers, including mechanisms of protein self-assembly. As noted above, production of this incredible fiber is accomplished under the mildest of conditions, at one atmosphere pressure and ambient temperature. Understanding how this is accomplished will undoubtedly have a significant impact on our ability to model and exploit biomimetic self-assembly processes that may yield materials with novel and desirable properties.
National Institutes of Health
Scalable Purification of Silk-Like Proteins From Transgenic Tobacco
South Carolina Agricultural Experiment Station
Structure-function Analysis of Group 1 LEA Proteins
Molecular Biology Laboratory
Gaines WA, Sehorn MG and Marcotte Jr WR. (2010). The spidroin N-terminal domain promotes a pH-dependent association of silk proteins during self-assembly. J Biol Chem, doi:10.1074/jbc.M110.163121
Gaines WA and Marcotte Jr WR. Recombinant dragline silk-like proteins-Expression and purification. AATCC Reviews, Accepted.
Manfre AJ, LaHatte G, Climer CR and Marcotte Jr WR (2009). Seed dehydration and the establishment of desiccation tolerance during seed maturation is altered in the Arabidopsis thaliana mutant atem6-1. Plant Cell Physiol, doi:10.1093/pcp/pcn185
Gaines IV WA and Marcotte Jr WR. (2008). Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes. Insect Mol Biol, doi: 10.1111/j.1365-2583.2008.00828.x
Teulé F, Marcotte Jr WR, Lewis, RV and Abbott AG (2008). Recombinant DNA methods applied to the production of protein-based biomaterials. In Biologically Inspired Textiles (A Abbott and M Ellison, eds.), Woodhead Publishing Limited, Cambridge, England. Pp. 3-25. ISBN 1845692470.
Gilles GJ, Hines KM, Manfre AJ1 and Marcotte Jr WR (2007). A predicted N-terminal helical domain of a Group 1 LEA protein is required for protection of enzyme activity from drying. Plant Physiol Biochem, 45:389-399.
Manfre AJ, Lanni LM and Marcotte Jr WR (2006). The Arabidopsis thaliana Group 1 LEA protein ATEM6 is required for normal seed development. Plant Physiol 140:140-149.