Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring Teeth Proteins Dawei Ding, †,‡ Paul A. Guerette, †,‡,§ Shawn Hoon, ∥,⊥ Kiat Whye Kong, ∥ Tobias Cornvik, ⊥ Martina Nilsson, ⊥ Akshita Kumar, ⊥ Julien Lescar, ⊥ and Ali Miserez* ,‡,⊥ ‡ School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798 § Energy Research Institute at Nanyang Technological University (ERI@N), Nanyang Technological University, 50 Nanyang Drive, Singapore, 637553 ∥ Molecular Engineering Lab, Biomedical Sciences Institute, A*STAR, 61 Biopolis Drive, Proteos, Singapore 138673 ⊥ School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 * S Supporting Information ABSTRACT: The sucker ring teeth (SRT) of Humboldt squid exhibit mechanical properties that rival those of robust engineered synthetic polymers. Remarkably, these properties are achieved without a mineral phase or covalent cross-links. Instead, SRT are exclusively made of silk-like proteins called “suckerins”, which assemble into nanoconfined β-sheet reinforced supramolecular networks. In this study, three streamlined strategies for full-length recombinant suckerin protein production and purification were developed. Recombi- nant suckerin exhibited high solubility and colloidal stability in aqueous-based solvents. In addition, the colloidal suspensions exhibited a concentration-dependent conformational switch, from random coil to β-sheet enriched structures. Our results demonstrate that recombinant suckerin can be produced in a facile manner in E. coli and processed from mild aqueous solutions into materials enriched in β-sheets. We suggest that recombinant suckerin-based materials offer potential for a range of biomedical and engineering applications. ■ INTRODUCTION In recent years, there has been a growing interest in structural biological materials produced by a diverse range of organisms. Structural, mechanical, and bioprocessing strategies of bio- logical systems are being investigated for the purpose of developing environmentally benign routes to synthesize novel materials. 1−4 Among recently investigated model organisms, cephalopods (squids, cuttlefish) have attracted interest in various areas of bioinspired engineering. For instance, squid have developed highly evolved sensory systems, 5 remarkable camouflage abilities, 6 fast and flexible, yet strong tentacles and arms, 7 as well as strong malleable suckers. 2 The Humboldt squid (Dosidicus gigas) is a large, aggressive, and predatory species that can be found in the Eastern Pacific ocean. These squid use two hard tissues in their predatory activities that have generated interest as potential biomimetic materials. The first is the tough, wear-resistant beak, which is used to lacerate tissues and subdue prey. The beak tip (rostrum) is one of the hardest and stiffest materials composed only of organic building blocks. 8 Its overall structure consists of a biomolecular composite made of hydrated chitin and Gly- and His-rich proteins. 9 These building blocks exhibit an opposing compositional gradient resulting in a mechanically graded material, with mechanical strengthening occurring through interprotein and protein-chitin covalent cross-linking. 10 The second load-bearing material of interest is found in the squid’s sucker ring teeth (SRT), which perform a grappling function in predation. Despite lacking a mineral phase, which is the common microstructural strategy used by Nature to make hard tissues, these structures display impressive mechanical proper- ties. 11,12 In contrast to the beak, SRT contain neither chitin nor interchain covalent cross-links and are instead entirely comprised of proteins called “suckerins”, which assemble into a supramolecular network reinforced by nanoconfined β-sheets that are embedded in an amorphous matrix. 2,13 Despite this unusual chemistry for a hard tissue, SRT exhibit mechanical properties that match those of strong synthetic polymers such as PMMA, PEEK, or polyamides. 14 At the molecular scale, SRT proteins (the most abundant being suckerin-39) display a regular modular sequence design 15 comprising two main types of alternating modules (Figure 1a,b). The first module is rich in alanine (Ala) and is reminiscent of poly-Ala β-sheet forming domains found in spidroins, 16,17 which reinforce spider dragline silk. 18 The second module is dominated by glycine (Gly) with a significant amount of tyrosine (Tyr) and leucine (Leu) residues organized as tri- and tetra-peptides, including GGY, GGL, or Received: May 9, 2014 Revised: July 1, 2014 Published: July 28, 2014 Article pubs.acs.org/Biomac © 2014 American Chemical Society 3278 dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289