International Journal of Biological Macromolecules 24 (1999) 265–270 Designing recombinant spider silk proteins to control assembly Stefan Winkler a , Sandra Szela a , Peter Avtges a , Regina Valluzzi a , Daniel A. Kirschner b , David Kaplan a, * a Tufts Uniersity, Biotechnology Center, Department of Chemical Engineering, Medford, MA 02155, USA b Boston College, Department of Biology, Chestnut Hill, Boston, MA 02467, USA Abstract The consensus repeat sequence found in the dragline silk from the spider, Nephila claipes, was redesigned to incorporate a redox trigger flanking the beta-sheet forming polyalanine sequences. The methionine redox trigger, in the oxidized state, was incorporated to prevent the formation of the beta sheets, while in the reduced state would not result in sterical limitations to beta sheet formation. A synthetic gene incorporating the trigger was constructed, cloned and then expressed in Escherichia coli. The purified protein, about 25 kDa, contained the expected amino acid composition and migration behavior on SDS-PAGE. The recombinant protein was analyzed by X-ray diffraction, TEM, electron diffraction and circular dichroism in both oxidized and reduced states. Based on the results, the incorporation of a redox trigger appears to be a powerful experimental strategy to explore the self-assembly of fibrous proteins such as silks. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Spider; Silk; Proteins; Assembly 1. Introduction Spider silks are some of the strongest natural fibers known and they rival high performance synthetic fibers in mechanical properties [1]. Strong natural fibers with good tensile and compressive properties would be use- ful for many applications in medicine, such as sutures, membranes and temporary scaffolds for tissue engineer- ing. To address this need, recombinant spider silks have been generated from native and synthetic spider silk genes using Escherichia coli as the heterologous host [2,3]. Upon purification, these recombinant silk proteins rapidly self-assemble to form insoluble microfibrils. From a purification point of view this is a useful property, however, it limits further processing of the protein into different types of fibers, films or coatings. Furthermore, we would like to understand the self-as- sembly process in detail to provide insight into control of this pathway. To resolve this problem, there is a need to regulate or control the assembly process so that appropriate concentrations and conformations of the protein can be maintained in solution and converted, on demand, to the final aqueous-insoluble silk form. To achieve this goal, ‘sterical-triggers’ are incorporated into the recombinant spider silks. Biosynthetic spider silk genes, which encode representative spider major ampullate gland silk, have been designed, constructed and expressed. These recombinant silks incorporate protein ‘triggers’ to control the solution behavior and assembly state of the silk proteins. 2. Material and methods 2.1. Strain and expression plasmids The E. coli strain BLR(DE3) ( F - ompT hsdS B ( r B - m B - ) gal dcm (srl -recA )306::TnlO (DE3)) (Novagen, Madison, WI), was used as a host for cloning and expression. The different silk gene constructs were cloned in the Bam H1 site of the expression vector pET29a (Novagen) [4], giving the construct pETnX, with X indicating the silk type and n representing the * Corresponding author. 0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII:S0141-8130(98)00088-9