Biomimetic Production of Silk-Like Recombinant Squid Sucker Ring

Jul 28, 2014 - ... Technological University, 60 Nanyang Drive, Singapore 637551 .... Multi-scale thermal stability of a hard thermoplastic protein-bas...
2 downloads 0 Views 6MB Size
Download PDF
Article pubs.acs.org/Biomac

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. Recombinant 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 biological 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 © 2014 American Chemical Society

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 properties.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 design15 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 3278

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Figure 1. Modular sequence design and recombinant expression of suckerin-39. (a) Large scale modular architecture of suckerin-39. Ala, Val, Thr, and His-rich modules are highlighted in red, Gly, Tyr, and Leu-rich modules are in blue. (b) Primary amino acid sequence and repetitive modular organization of suckerin-39 (signal peptide is underlined; red and blue modules are highlighted as described in (a), and proline residues are highlighted in green). (c) SDS-PAGE of expression products of His-suckerin-39 and suckerin-39. MW, molecular weight; M, molecular weight markers; S1 and P1, supernatant and pellet from cells expressing His-suckerin-39, respectively; S2 and P2, supernatant and pellet from cells expressing suckerin-39, respectively. (d) Reconcentrated elution fractions of His-suckerin-39 purified by IMAC with 1 h binding at room temperature (E1), 1 h binding at 4 °C (E2), and 15 h binding at 4 °C (E3); (e) MALDI-ToF spectra of His-suckerin-39 purified by IMAC at different conditions (E1, E2, and E3 fractions shown in (b)).

GC-rich silk gene sequences are often unstable in E. coli. Codon biases, tRNA pool depletion, homologous recombination, segmental deletions and expansions, transcription errors, unusual mRNA structure, truncated synthesis, translational pausing, and protein aggregation may all limit the efficiency of silk production in E. coli.25−30 Therefore, a wide range of systems, including yeast, plants, mammalian cell lines, and transgenic organisms, have also been evaluated for the generation of recombinant silk proteins, each of which has met with varying degrees of success.25,30−32 Among the most significant advances, Xia et al.33 have reported high yield (1.2 g/ L from high cell density cultivation) of a 285 kDa dragline silk protein mimetic produced in metabolically engineered E. coli cells where recombinant proteins were spun into fibers whose mechanical properties approached those of the native materials. A second major goal in silk biotechnology is to develop efficient methods for the solubilization and processing of native and recombinant silk proteins. These proteins have a wellknown tendency to prematurely adopt β-sheet structures and aggregate under aqueous-based conditions. While an impressive range of structures and functional materials have been generated using both native silkworm fibroins and recombinant silk-like proteins,34,35 many published processing strategies involve the use of harsh conditions, including 9 M lithium bromide (LiBr) and hexafluoroisopropanol (HFIP) to solubi-

GGLY. The similarity between suckerins and silk proteins is appealing given the wide range of biomedical and engineering applications demonstrated for silks, including in tissue engineering,19 drug delivery,20 and photonics.21 We previously demonstrated that suckerins exhibit similar potential, where native and recombinant suckerins were rapidly processed from concentrated aqueous solutions into various shapes and functional materials.15 In addition, as is the case for silk proteins, suckerins are likely to be readily amenable to functionalization by genetic engineering22,23 and chemical modification.24 Silk science continues to evolve at a rapid pace and provides an important benchmark for the evaluation of newly discovered load bearing proteins such as the suckerins. Over the past decade there have been significant efforts to achieve high yield recombinant expression of silk proteins and to establish streamlined green processing protocols that maintain silk protein solubility with minimal usage of harsh and toxic solvents. These two goals have met with several major challenges. Because of its ease of manipulation, short generation time, well-understood genetics, and availability of established expression vectors, E. coli is the most commonly used host for recombinant silk protein production. E. coli is established as a suitable host for the industrial scale production of a wide range of recombinant proteins and offers excellent flexibility for industrial-scale production.25 However, the highly repetitive, 3279

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

His-tagged and nontagged suckerin constructs were verified by Sanger sequencing. Expression of His-Suckerin-39 and Suckerin-39. A 10 μL aliquot of cells from each glycerol stock were streaked onto LB agar plates containing kanamycin (30 μg/mL) and ampicillin (100 μg/mL), respectively, and grown overnight at 37 °C. Single colonies were inoculated into selective LB media (30 μg/mL kanamycin and 100 μg/ mL ampicillin for His-suckerin-39 and suckerin-39 cells, respectively) for overnight shaking cultures at 37 °C. The next day, stationary phase cell cultures were diluted 100-fold into fresh LB media (with the same concentrations of antibiotics) and grown under the same conditions until an OD600 of ∼0.6−1 was achieved. Protein expression was induced by adding 0.5 mM IPTG and cultures were further incubated with shaking for 3−4 h before harvesting by centrifugation at 14000 g for 10 min and frozen at −20 °C. Purification of His-Suckerin-39 by Immobilized Metal Affinity Chromatography (IMAC). Cell pellets were resuspended in lysis buffer (20 mM Tris-HCl, pH 7.4), lysed by sonication (40% amplitude, 1 s pulse + 1 s pause, 7.5 min sonication), and clarified by centrifugation at 23000 g for 10 min at 4 °C. The pellet was resuspended in 8 M urea and 5% acetic acid at 4 mL per gram wet weight of pellet. Small aliquots (10 μL) of both supernatants and ureasolubilized pellets were loaded into SDS-PAGE gels to verify the protein expression for each cell line. His-suckerin-39 was purified from inclusion bodies under denaturing conditions by IMAC. His-suckerin39 inclusion bodies were dissolved in a solution of 5% acetic acid with 8 M urea, and 0.5 mL of this solution was mixed with 7.5 mL of binding buffer (8 M urea, 20 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 7.8). The sample was then incubated with 1 mL NiNTA resin (Qiagen), allowing the binding of proteins to the resin. Two parameters were evaluated, namely, the binding temperature (4 °C and room temperature) and binding time (1 and 15 h). After binding, the flow-through from the column was collected and the resin was rinsed twice with 5 mL of wash buffer (8 M urea, 20 mM NaH2PO4, 0.5 M NaCl, 30 mM imidazole, pH 7.8). Finally, the protein was eluted from the resin with 10 mL of elution buffer (8 M urea, 20 mM NaH2PO4, 0.5 M NaCl, 500 mM imidazole, pH 7.8). The samples were then dialyzed against 5% acetic acid, freeze-dried, solubilized in 0.5 mL 5% acetic acid, and subjected to SDS-PAGE analysis. Their purity and molecular weights were further confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDIToF) mass spectrometry (AXIMA-TOF2, Shimazu) with Sinapinic acid (Sigma) dissolved in a mixture of 50/50 Q-water/ACN with 0.1% TFA as the matrix. Purification of His-Suckerin-39 and Suckerin-39 by SaltingOut. Cell pellets were lysed as described above and His-suckerin-39 was purified by salting-out using a buffer with a pH close to its calculated isoelectric point (IEP) (8 M urea, 20 mM NaH2PO4, 0.5 M NaCl, 10 mM imidazole, pH 7.8). Inclusion bodies were solubilized in 8 M urea and 5% acetic acid at 4 mL solution/gram of inclusion bodies and dialyzed twice against the salting out buffer at a volume ratio of 1:1000. The precipitated proteins were collected by centrifugation at 16000 g for 10 min and the pellet was resolubilized in 5% acetic acid. Suckerin-39 was purified with a similar salting-out method, where the pH of the salting-out was adjusted to 8.4 to account for the higher IEP of non-His-tagged suckerin. Purification of Suckerin-39 by Microfluidization, Followed by Inclusion Body Purification. Harvested cell pellets were washed twice with lysis buffer (20 mM Tris, pH 8). Each pellet from 1 L culture was resuspended in 50 mL of lysis buffer (50 mM Tris, pH 7.4, 200 mM NaCl, 1 mM PMSF) and lysed by six passes of microfluidization (Microfluidics M-110P). The lysate was clarified by centrifugation at 52000 g for 1 h at 4 °C. The pellet was washed twice with 20 mL wash buffer 1 (100 mM Tris, 5 mM EDTA, 2 M urea, 2% (v/v) Triton X-100, 5 mM DTT, pH 7.4) followed by two washes of wash buffer 2 (100 mM Tris, 5 mM EDTA, 5 mM DTT, pH 7.4). The inclusion bodies were collected by low speed centrifugation (5000 g for 15 min at 4 °C) between wash steps. Finally, the pellet was resolubilized in 5% acetic acid.

lize and process these proteins. However, recent studies have focused on processing fibroins from the aqueous phase under more environmentally friendly conditions, with promising results.35,36 Gram quantities of native SRT can be collected for laboratory-scale experiments. However, large-scale production of suckerin for biomedical and engineering applications will require a reliable source of recombinant protein. In this study we first aimed to test and establish streamlined recombinant expression and purification strategies for suckerins, which will eventually be suitable for large-scale production. Second, we aimed to establish the conditions that maintain solubility of recombinant suckerins and to compare the behavior of suckerin in solution with that of silk proteins. Two versions of full-length suckerin-39 were expressed in E. coli. These were (i) a codonoptimized, poly-His tagged suckerin-39, and (ii) a nontagged native coding sequence of the full-length suckerin-39. Fulllength recombinant suckerin proteins were readily expressed in inclusion bodies and easily purified by chromatographic and nonchromatographic methods. For the His-tagged suckerin, following immobilized metal affinity chromatography (IMAC) purification, the His-tag was removed by TEV protease cleavage and further purified by ion exchange fast protein liquid chromatography (FPLC). For the native suckerin-39, purification was achieved by a straightforward salting-out procedure (iso-electric precipitation) or by stringent washing of inclusion bodies, followed by solubilization. Our data show that, in contrast to many reports on recombinant silk protein production, the suckerin genes are stable in bacterial systems. Recombinant suckerin-39 was found to exhibit high solubility in aqueous solutions with values up to 70.9 mg/mL in mild acidic environments. In water alone, the solubility was 7.2 mg/mL. Solubilized suckerins were found to assemble into colloids of discrete dimensions, which are suggested to offer a route toward the facile processing of these proteins into hierarchical structures. The results establish relevant parameters for the processing of suckerin-based materials from aqueous solutions and for the green processing of high-performance “bioplastics”.

■

EXPERIMENTAL SECTION

Cloning of His-Suckerin-39 and Suckerin-39. The full-length open reading frame (ORF) of suckerin-39 was originally obtained from the transcriptome assembly of the Humboldt squid sucker tissue15 and confirmed by RACE-PCR. The codon-optimized gene encoding suckerin-39 was purchased from DNA 2.0 (Menlo Park, California). A PCR product containing this gene was then generated using forward primers that included LIC cloning sequences: Hissuckerin-39-FWD primer: 5′-TGTGAGCGGATAACAATTCC-3′; His-suckerin-39-REV primer: 5′-AGCAGCCAACTCAGCTTCC-3′. The PCR product was isolated using a QIAquick Gel Extraction Kit (Qiagen) and cloned into the LIC sites of the pNIC-28-BsaIV plasmid vector that included sequences encoding a 5′-poly-His tag and a linker peptide containing a TEV protease cleavage site. The plasmid constructs were then transformed into E. coli Rosetta cells. For nontagged suckerin-39, the expression cell line was previously established.15 Briefly, the full-length ORF omitting the signal peptide sequence was amplified by RT-PCR using KOD polymerase and the following primers: suckerin-39-FWD primer: 5′-AAAAAAGCTAGCATTTTGCCAGCGGCAACATCTG-3′;suckerin-39-REV primer: 5′AAAAAACTCGAGTTAGTGGAGGAGACCATATCCAC-3′. The PCR product was then purified using a QIAquick Gel Extraction Kit (Qiagen) and cloned into the into the NheI/XhoI site of a pET23a vector (Novagen), which was transformed into E. coli BL21 (DE3) cells. Cell stocks were made in 20% glycerol. The sequences of both 3280

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Screening of Buffers to Cleave the His-Tag from HisSuckerin-39 Using TEV Protease. A solubility improved variant of recombinant TEV protease was generated according to published methods37 (the TEV protease clone was kindly provided by Dr. Helena Nerglund, Karolinska Institute). In order to cleave the His-tag from the recombinant His-suckerin-39, various buffer conditions were screened, with the goal of keeping His-suckerin-39 soluble while maintaining TEV protease activity. Buffers containing 0.5 mM EDTA, 1 mM DTT, and different concentrations of urea from pH 4.0 to 9.0 were screened. Eluted fractions of His-suckerin-39 with concentrations ranging from 0.1 to 0.2 mg/mL were dialyzed against these buffers overnight and centrifuged at 16000 g for 5 min to spin down possible precipitates. Supernatants and pellets (dissolved in 5% acetic acid) were analyzed by SDS-PAGE. In addition, the enzymatic activity of TEV protease in each buffer was evaluated using a SUMO fusion protein, which contained an engineered TEV protease cleavage site.38 Buffers were considered not useful if His-suckerin-39 precipitation occurred or if TEV protease did not cleave the SUMO fusion protein in test digestion experiments, which involved verifying the presence of two bands by SDS-PAGE in the case of successful cleavage. Evaluation of Solubility of Suckerin-39 in Different Solutions. Freeze-dried suckerin-39 was dissolved in different solutions including milli-Q water, 5% acetic acid (v/v), and 8 M urea containing 5% acetic acid (v/v). In order to measure the maximum solubility of suckerin-39 in each solution, small volumes of solution were sequentially added to the suckerin-39 powder until the majority of the proteins was completely dissolved. A total of 5 min of sonication was used to facilitate the dispersion. The suspension was then centrifuged for 5 min at 16000 g to remove the minor fraction of insoluble products and the protein concentration of each supernatant was analyzed with the Bradford protein assay kit (Thermo Scientific). Each sample was diluted to below 1 mg/mL with respective solvent before the measurement. The maximum protein solubility was measured 3 to 4 times for each solvent tested and calculated using a standard curve. We also evaluated the solubility and refolding/assembly behavior of suckerin-39 in several buffered saline solutions (Table S2). For these experiments, 0.1 mg/mL of 5% acetic acid solubilized protein was dialyzed against each buffer. The dialysate was then centrifuged at 16000 g for 15 min. The supernatant was concentrated from an initial volume of 1 mL to a volume of ∼100 μL with a Vivaspin Sample Concentrator (GE Healthcare) and the pellet was dissolved in an equivalent volume of 5% acetic acid. Both fractions were then evaluated by SDS-PAGE to infer solubility/insolubility (data not shown) and suckerin concentrations in the supernatant fractions were quantified using the Bradford assay as described above. Biophysical Characterization of Suckerin-39. Particle dimensions of suckerin-39 were determined by dynamic light scattering (DLS). Suckerin-39 was resuspended in 5% acetic acid and water at 1 and 6 mg/mL or in 0.05 M MES (2-[N-morpholino]ethanesulfonic acid), pH 5.5, at 0.64 mg/mL, and the samples were analyzed with a 90Plus particle size analyzer (Brookhaven Instruments) equipped with a 658.0 nm monochromatic laser. To minimize the reflection effect, all measurements were taken at a scattering angle of 90°. The numberweighted histogram profiles of the proteins in solution were used for data analysis. The mean particle size of suckerin-39 colloids in water was also investigated by the atomic force microscopy (AFM; MFP-3DBIO, Asylum Research). The protein was diluted to 10 μg/mL with water and single drops of the solution were placed on mica substrates, and allowed to equilibrate for 20 min followed by rinsing with water. Finally, the sample was air-dried and subjected to AFM imaging. The mean size of particles from three images was measured by ImageJ. The zeta-potentials of these samples were measured in triplicate at 25 °C with 15 scans for each measurement using a Nano-ZS Zeta sizer (Malvern). The secondary structure of suckerin-39 was characterized by circular dichroism (CD) using a Chriascan spectropolarimeter (Model 420, AVIV Biomedical Inc.). A total of 1 and 6 mg/mL of suckerin-39 in water and in 5% acetic acid, and 0.64 mg/mL suckerin-39 in 0.05 M MES at pH 5.5 were used for the measurements, which were

conducted in triplicate at wavelengths ranging from 190 to 260 nm, with 1 nm step size and 1 nm bandwidth. The spectra were smoothed by the Savitzky-Golay method with a polynomial order of 2. The secondary structure of suckerin-39 in the various buffers was also measured by Fourier transform infrared (FTIR) spectroscopy. A Vertex 70 instrument (Bruker) was employed in the Attenuated Total Reflection (ATR) mode, with 4 min scanning and a resolution of 4 cm−1 in the 4000−400 cm−1 region. The respective solvents were used as the controls. The protein concentration was 6 mg/mL for 5% acetic acid and water-solubilized samples and 0.64 mg/mL for 0.05 M MES (pH 5.5) solubilized samples. In order to estimate the fraction of different secondary structures, the amide I band was deconvoluted using OPUS 6.5 software (Bruker). The positions of the fitting peaks were fixed based on peaks identified in the second derivative of amide I. In this case, the widths of the fitted peaks were approximately the same.

■

RESULTS AND DISCUSSION Expression of His-Suckerin-39 and Suckerin-39. Two types of E. coli cells, Rosetta cells, and BL21 (DE3) cells were cultured for suckerin expression. Both His-suckerin-39 and suckerin-39 were almost exclusively found in the pellet after cell lysis (Figure 1c), demonstrating that these proteins were predominantly assembled into inclusion bodies during expression. The appearance of inclusion bodies is usually explained by protein overexpression that can result in protein misfolding and formation of insoluble aggregates. While misfolding and the formation of inclusion bodies is usually problematic for downstream applications, it is advantageous in the context of protein-based biomaterials because it provides an efficient preseparation of relatively pure target proteins from the bacterial cytoplasmic proteins. As seen in Figure 1c, the inclusion bodies contained a relatively low number of proteins. Purification of His-Suckerin-39 by IMAC. Urea-solubilized His-suckerin-39 was bound to Ni-NTA resin under varying conditions (1 h at room temperature, 1 h at 4 °C, and 15 h at 4 °C) and eluted under denaturing conditions. The elution fractions were subsequently dialyzed against 5% acetic acid, freeze-dried, and reconstituted in 5% acetic acid. All three binding conditions resulted in pure His-suckerin-39 preparations, as shown by SDS-PAGE analysis (Figure 1d). A total of 15 h of binding at 4 °C was identified as optimal for Hissuckerin-39 IMAC binding. The purity and molecular weight of His-suckerin-39 were then evaluated by Maldi-ToF. The elution fractions exhibited a major peak at around 42 kDa (Figure 1e), corresponding to the expected MW of suckerin-39 (39.4 kDa) with His-tag and linker peptide (2.7 kDa). Doubly and triply charged species were also detected. A few low molecular weight impurities were observed, but their intensity was extremely low. These data demonstrate that His-suckerin39 purification by IMAC is straightforward. However, the presence of the His-tag may be problematic for some applications and for subsequent self-assembly. Cleavage of His-Tag with TEV Protease. While affinity tags can provide several benefits to recombinant proteins, such as facilitating purification, increasing yield and solubility, and even promoting the folding of target proteins,39 they have also been reported to negatively affect the target proteins. For instance, they can affect protein conformation, alter the biological activity, lead to undesired flexibility in structural studies, and introduce toxicity.40 Therefore, we established protocols for the removal of the tag from His-suckerin-39. TEV protease was expressed in E. coli and purified with established protocols.37 Recombinant TEV protease activity 3281

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Figure 2. Cleavage of His-tag from His-suckerin-39. (a) Products of His-suckerin-39 in IMAC elution buffer dialyzed against buffers with different pH (S, supernatant; P, pellet); (b) Cleavage of SUMO fusion protein by TEV protease in the buffers used in (a); (c) Time-dependent His-tag cleavage from His-suckerin-39 from 2 to 24 h; (d) Ion exchange-FPLC profile of detagged His-suckerin-39. The inset shows the SDS-PAGE analysis of the detagged His-suckerin-39 peak: Fractions 35−40; (e) Maldi-ToF spectrum of detagged His-suckerin-39 after ion exchange FPLC purification.

activity were MES buffer with 2 M urea at pH 5.5 (fully soluble) and pH 6 (partially soluble), as illustrated in Figure 2a,b. These conditions were then used to cleave the His-tag of His-suckerin-39. The results revealed that cleavage was timedependent. Approximately half of the His-tags were removed within 2 h, and the cleavage continued until the His-tags were totally removed by TEV protease after 24 h at room temperature (Figure 2c), giving rise to detagged His-suckerin39. The cleaved His-suckerin-39 was separated from TEV protease by ion exchange FPLC, where two distinct peaks were eluted from the column at around 37 and 43 mL (Figure 2d). SDS-PAGE analysis revealed that the first peak corresponded to pure detagged His-suckerin-39 (Figure 2d inset), whereas the second peak was TEV protease (SDS-PAGE data not shown). We found it was challenging to purify detagged His-suckerin-39 from TEV protease by IMAC although the later had a polyHistidine tag. This may be due to the high His content of suckerin-39,15 making it prone to bind to Ni-NTA resin and to be eluted together with TEV protease. The molecular weight of detagged suckerin-39 was confirmed by MALDI-ToF (Figure 2e), with a peak at 39.4 kDa matching the expected MW. Purification of Suckerin-39 by Salting-Out. In addition to IMAC purification, simpler nonchromatographic methods for His-suckerin-39 and suckerin-39 purification were also established. We noticed that His-suckerin-39 tended to precipitate when their 5% acetic acid/8 M urea solubilized

was evaluated using the established model SUMO fusion protein. After incubation of the SUMO protein with TEV protease in standard cleavage buffer, two bands at ∼20 and 25 kDa appeared after 2 h and the cleavage was completed after 4 h (data not shown), indicating that TEV protease was active and suitable for cleavage of the His-suckerin-39 tag. In order to cleave the His-tag, we screened a series of buffers to establish conditions where His-suckerin-39 was soluble and TEV protease was active. Any conditions that failed to fulfill these two requirements were eliminated and the results are summarized in Table S1. The standard working buffer of TEV protease is 50 mM Tris-HCl containing 0.5 mM EDTA and 1 mM DTT (pH 8.0) and its activity is not decreased at a pH between 4 and 9.41 Therefore, we first investigated the solubility of His-suckerin-39 in buffers ranging from pH 4.0 to pH 9.0 containing 50 mM sodium acetate, MES, or Tris-HCl, according to the enzyme’s compatibility.41 His-suckerin-39 was not soluble in any of these buffers and we found that a minimum of 2 M urea was required to maintain solubility in these buffers. Under such denaturing conditions, His-suckerin39 precipitated at pH 6.5 and above. It has been reported that TEV protease can tolerate high concentrations of denaturants, such as urea, up to 4 M.42 We verified TEV protease activity with 2 M urea and found that it remained active at pH > 5. In summary, this screening study established that suitable conditions for His-suckerin-39 solubility and TEV protease 3282

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

higher IEP of 8.4 (compared to 7.6 for the His-tagged version), the same buffer adjusted to pH 8.4 was used for the dialysis. As shown by SDS-PAGE and MALDI-ToF analysis (Figure 3b), high purity suckerin-39 was obtained after dialysis. In contrast to IMAC purification, this method was more efficient, eliminating the need for His-tag removal and requiring fewer steps and reagents. Purification of Suckerin-39 by Microfluidization and Inclusion Body Purification. During the purification of recombinant proteins from inclusion bodies, it is important that cells are lysed completely to reduce the amount of contaminating proteins. Additional methods for suckerin-39 inclusion body purification were therefore developed. We found that cell lysis by microfluidization was both high throughput and effective for this goal. Cells collected after culture were resuspended in lysis buffer and passed through a microfluidizer 6× in order to completely disrupt the cells and release the inclusion bodies. After microfluidization, suckerin-39 remained mostly in the insoluble pellet. The pellet was washed twice with buffers containing 2 M urea and 2% Triton X-100 to remove cellular membranes and other contaminating proteins (Figure 3c, lanes W1.1 and W1.2). This was followed by two detergentfree washes to remove the Triton X-100 and low-molecular weight contaminants (Figure 3c, left, lanes W2.1 and W2.2). Very little suckerin-39 loss was observed. Suckerin-39 was obtained by resolubilizing the pellet in 5% acetic acid, which resulted in sample purity comparable to IMAC purification, where MALDI-ToF indicated the presence of a small quantity of low molecular weight contaminants (Figure 3c, right). The yields of suckerin-39 for each purification method are listed in Table 1. Yields of recombinant silk proteins produced in various hosts are provided for comparison. A total of 4.3 mg of protein per liter of culture was obtained by the IMAC method for His-tagged suckerin-39, while the yield of nontagged suckerin-39 by salting-out was 5.9 mg per liter of culture. By microfluidization, the yield was 20.2 mg/L of cell culture. Of the three methods tested, microfluidization proved to significantly improve yield and efficiency. While our saltingout method required excessive amounts of urea that would not be suitable for scale-up, the current microfluidization purification methodology required only 40 mL of 2 M urea per liter of culture, which we consider a reasonable step forward. The apparent ease of production of recombinant suckerin-39 in E. coli likely relates to its manageable size and lower degree of highly repetitive sequences compared to silk proteins. No attempts were made to optimize the expression conditions, such that significant opportunities remain to further increase the yield. Future studies in our lab will involve a direct evaluation of the performance of Rosetta cells compared to BL21 (DE3) cells and the quantitative evaluation of yields from codon optimized and native suckerin genes. We will also conduct a systematic optimization of expression yields as has been done for the efficient expression of silk-elastin like peptides (SELPs) in E. coli.47 SELPs, which can have similar molecular weights to suckerin-39, exhibit excellent aqueousbased solubility and have gained strong traction in the biomimetic engineering of robust functional materials.47−50 We anticipate that expression yields as high as those obtained for silk and SELP constructs should be attainable for suckerins. Solubility of Suckerin-39. The processing of proteins at high concentrations is desirable for several reasons. Natural secretions are often processed at high concentrations, and

inclusion body fractions were dialyzed against the IMAC binding buffer at pH 7.8. Following centrifugation, we observed that the pellet contained His-suckerin-39 at relatively high purity (Figure 3a, left). This result is not surprising given that

Figure 3. Purification of His-suckerin-39 and suckerin-39. (a) Saltingout products of His-suckerin-39 in IMAC binding buffer at pH 7.8 (left) and corresponding MALDI-ToF spectrum of the pellet (right). S and P: supernatant and pellet after dialysis, respectively. (b) Saltingout products of suckerin-39 in IMAC binding buffer at pH 8.4 (left) and corresponding MALDI-ToF spectrum of the pellet (right). (c) Purification of suckerin-39 by microfluidization and corresponding MALDI-ToF spectrum of the pellet (right). S: supernatant after cells lysis with a microfluidizer; W1.1, W1.2: supernatant washing fractions from wash buffer 1; W2.1, W2.2: supernatant washing fractions from wash buffer 2; 5% AA: Suckerin-39 solubilized by 5% acetic acid after washing steps.

the pH of the binding buffer was close to the theoretical isoelectric point of His-Suckerin-39 (calcd pI = 7.6, based on the full sequence, including the poly-His tag and linking peptide), leading to a protein net charge close to zero, thus, facilitating its isoelectric precipitation. Notably, His-suckerin-39 aggregated despite the presence of 8 M urea. The high purity was confirmed by MALDI-ToF analyses, which indicated peaks at around 14, 21, and 42 kDa corresponding to the triply, doubly, and singly charged His-suckerin-39, respectively (Figure 3a, right). From these observations, this salting-out method was evaluated with nontagged suckerin-39. Based on its 3283

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Table 1. Summary of Recombinantly Expressed His-Suckerin-39, Suckerin-39, and Spider Silk Proteins in Different Hostsa protein His-suckerin-39 suckerin-39 suckerin-39 MaSp-128 MaSp-133 MaSp-143 MaSp-144 MaSp-145 ADF-346

origin Dosidicus gigas Dosidicus gigas Dosidicus gigas Nephila clavipes Nephila clavipes Nephila clavipes Nephila clavipes Nephila clavipes Araneus diadematus

host

MW (kDa)

E. coli E. coli E. coli E. coli E. coli P. pastoris B. mori N. tobaccum BHK cells

42 39 39 65−163 100−285 65 70 13−100 60−140

purification strategy IMAC salting-out microfluidization IMAC ammonium sulfate ammonium sulfate IMAC ammonium sulfate ammonium sulfate

yield (mg L−1) b

4.3 5.9 20.2 300 1200c 663 6 mg/larva 0.5−2.0% total protein 25−50

a

Recombinantly expressed spider silk proteins with highest yield in each host are shown. bYield before TEV protease cleavage. cHigh cell density cultivation (HCDC) of the engineered cells was performed to achieve high yield of proteins.

Figure 4. (a) Solubility limit of suckerin-39 in different solutions. The inset shows suckerin-39 concentration in log scale (AA: acetic acid). (b) Particle size distribution of suckerin-39 in 5% acetic acid and water at 1 mg/mL and in 0.05 M MES, pH 5.5, at 0.64 mg/mL. (c) Particle size distribution of suckerin-39 colloids solubilized in water determined by AFM in the dry state. The inset shows a representative AFM image of suckerin-39 colloidal particles assembled in water and viewed in the dry state. (d) DLS particle size distribution of suckerin-39 colloids in 5% acetic acid and water after 2 weeks at 4 °C.

(Figure 4a). In contrast, suckerin-39 exhibited a much higher maximum solubility in 5% acetic acid/8 M urea (85.1 mg/mL). Interestingly, the solubility limit of suckerin-39 in 5% acetic acid (70.9 mg/mL) was close to that in 5% acetic acid/8 M urea and about 10-fold greater compared to water. Suckerin-39 precipitated in all buffered saline solutions tested (Table S2); however, it remained soluble up to 0.64 mg/mL in 0.05 M MES at pH 5.5. Given that high protein concentration is often a requirement to produce protein-based biomaterials,35,51 the high solubility of suckerin-39 in acetic acid will likely be critical for the fabrication of bulk materials. Furthermore, 5% acetic acid has been shown to be a promising solution for film fabrication of suckerin-39 from initial protein concentrations of ∼20 mg/mL.15 Hence, the higher solubility limit under mild

molecular crowding may facilitate their hierarchical assembly into bulk materials. In addition, processing from dilute preparations can require excessive solvents and thus impact scalability. Native and recombinant silk fibroin proteins generally require harsh solvents to maintain solubility at elevated concentrations where optimal working concentrations for recombinant silk fibroin preparations are usually in the 10− 80 mg/mL range. In order to explore the potential for fabrication of suckerin-39-based materials from concentrated solutions, particularly using mild processing strategies, we investigated the solubility limit of suckerin-39 in different aqueous solutions, including water, 5% acetic acid, 5% acetic acid/8 M urea, and various buffered saline solutions. Suckerin39 exhibited a maximum solubility of 7.2 mg/mL in water 3284

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

conditions provides an additional degree of versatility that may enable tuning of the properties of suckerin-39-based biomaterials by adjusting the initial protein concentration. While the underlying reason for the high solubility of suckerin-39 in mild acidic conditions has not been explored, the high His content (9.8 mol %) and absence of other charged residues in suckerin-39 are likely to play a critical role. Below the pKa of His (6.5), suckerin-39 bears a net positive charge due to protonation of the imidazole moieties, resulting in electrostatic repulsion of individual chains and increased protein solubility, as also confirmed by zeta potential measurements discussed in the next Section. Biophysical Characterization of Suckerin-39. Biophysical characterizations were performed on purified suckerin-39 in order to investigate the influence of aqueous-based environments on assembly and secondary structure. In acidic conditions (5% acetic acid) and at low protein concentration (1 mg/mL), DLS measurements indicated that suckerin-39 existed in a monomeric state, with a mean hydrodynamic diameter DH of about 5 nm (Figure 4b and Table 2). At an Table 2. Particle Diameter and Zeta-Potential of Suckerin-39 particle diameter (nm)

solvent

concentration (mg/mL)

5% acetic acid

1.0

6±2

H2O

6.0 40.0 1.0 6.0 0.64

63 94 69 80 52

0.05 M MES

± ± ± ± ±

22 10 7 7 2

polydispersity indices

zetapotential (mV)

0.31 ± 0.02

43.2 ± 1.8

0.30 0.30 0.31 0.30 0.24

± ± ± ± ±

0.01 0.02 0.01 0.01 0.02

30.3 ± N.A. 44.2 ± 33.6 ± 19.7 ±

0.6 0.2 0.6 0.4

increased concentration of 6 mg/mL, DH increased by 10-fold to ∼60 nm (±22 nm), suggesting the formation of suckerin-39 oligomers. When solubilized in water (∼pH 6.6), the hydrodynamic diameter distribution ranged from 60 to 90 nm at both 1 and 6 mg/mL (Figure 4b and Table 2) with an average at around 70 and 80 nm, respectively, suggesting that suckerin-39 oligomers are stable in water up to their solubility limit. In 0.05 M MES at pH 5.5, 0.64 mg/mL suckerin-39 exhibited a DH distribution around 50 nm (Figure 4b and Table 2). The polydispersity indices of suckerin-39 in 5% acetic acid, water, and 0.05 M MES were similar (around 0.30) at all concentrations tested (Table 2). The particle size of suckerin-39 colloids assembled in water was further investigated by AFM, where globular particles ranging from 15 to 80 nm in diameter were observed on the substrate (Figure 4c, inset). In this case, the particles exhibited a bimodal size distribution, with one group exhibiting a mean diameter of ∼20−30 nm and the other a mean diameter of ∼50−60 nm (Figure 4c). We do not consider the mean particle diameters measured by AFM imaging to be a direct reflection of the colloidal particles observed in solution since the sample had to be diluted for imaging and given that the substrate may also influence assembly, potentially affecting the oligomerization state of the particles. Therefore, the DLS and AFM data should not be directly compared. Circular dichroism (CD) was then used to evaluate the secondary structures of suckerin-39 in 5% acetic acid, water, and 0.05 M MES, pH 5.5 (Figure 5). In 5% acetic acid and milli-Q water, a conformational switch was observed with

Figure 5. Secondary structures of suckerin-39 in solution. (a) CD spectra of suckerin-39 at different concentrations in 5% acetic acid; (b) CD spectra of suckerin-39 at different concentrations in water; (c) CD spectrum of 0.64 mg/mL suckerin-39 in 0.05 M MES, pH 5.5.

increased concentration. At 1 mg/mL, the CD signature corresponded to predominantly random-coil structure with a minimum at around 195−200 nm.52 On the other hand, at higher concentration (6 mg/mL), suckerin-39 exhibited a minimum at 215 nm and a maximum at 198 nm, which is characteristic of β-sheet structure.52 A β-sheet signature was also observed for 0.05 M MES solubilized suckerin-39 with a minimum at 218 nm and a maximum at 202 nm (Figure 5c; Standard CD curve fitting software did not provide reasonable fits for % structure estimates of the data presented in Figure 5 and we restrict our interpretation qualitative observations in this case). Taken together, the data suggest that suckerin-39 can be refolded into β-sheet enriched structures in aqueous-based solutions, with a secondary structure bearing similarity with the native structure of SRT.15 The occurrence of β-sheets within 5% acetic acid, water and 0.05 M MES (pH 5.5) solubilized suckerin-39 preparations was supported by FTIR (Figure 6). The spectra were characterized by strong absorbances at ∼1623−41 cm−1 assigned to β-sheet 3285

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Figure 6. ATR-FTIR analysis of recombinant suckerin-39. (a) Full FTIR spectra of suckerin-39 in 5% acetic acid (6 mg/mL), water (6 mg/mL), and 0.05 M MES, pH 5.5 (0.64 mg/mL). (b−d) Deconvoluted amide I spectra suckerin-39 FTIR spectrum for the samples respectively described in (a) . The positions of fitting peaks were defined by the 2nd derivative of the amide I spectra and the width of each peak was fixed.

Figure 7. Schematic of oligomerization state and structure of suckerin-39 in 5% acetic acid, water, and 0.05 M MES, pH 5.5, at different concentrations.

3286

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

Table 3. Comparison between SRT and Silkworm or Spider Dragline Silks properties native morphology native mechanical loading regime mechanical properties structure protein composition molecular weight primary amino acid sequence ease of full-length recombinant protein expression ease of processing and fabricating materials engineered materials

SRT

silkworm and spider dragline silks

bulk tensile, compression, shear15

fiber tensile57

high E (4.5−8 GPa)2,15 β-sheet reinforced network15 suckerins15 8−50 kDa2,15 Ala, Val, Thr, Ser, His-rich, and Gly, Tyr, Leu-rich modules15 yes15

high E (∼10 GPa)57−59 β-sheet reinforced network18,57 fibroins or spidroins35,57 heavy chain silk fibroin, 390 kDa; light chain silk fibroin, 26 kDa; spidroins, 250−320 kDa60,61 poly(Gly-Ala)/poly-Ala and Gly-rich modules57

rapid purification, facile waterbased fabrication15 fibers, films, nanostructures, nanospheres, tissue scaffolds15

harsh solvents often required for purification and fabrication35

no25,31

fibers, films, nanofibers, microspheres, sponges, hydrogels, tissue-scaffolds35,62

structures of the amide I region, as well as by peaks at ∼1520 cm−1 corresponding to β-sheet conformation in the amide II region.53 In addition, peaks at ∼1242 cm−1 attributed to β-sheet structure were also observed in the amide III region (Figure 6a).54 Semiquantitative analysis of the secondary structures of suckerin-39 were obtained by standard peak deconvolution55 of the amide I band. As shown in Figures 6b−d, Suckerin-39 exhibited a relatively high content of β-sheet conformations in 5% acetic acid (26.2%), water (34.7%), and 0.05 M MES at pH 5.5 (42.7%). Other conformations were generally less abundant, including helix, random coil, and β-turns.55,56 The formation of oligomers coupled with the detection of β-sheets, schematically represented in Figure 7 for the various conditions tested, suggests that oligomerization is enabled by the formation of interchain β-sheets at higher protein concentrations. We also measured the zeta-potential of suckerin-39 in different solutions. As shown in Table 2, the zeta potential of suckerin-39 in 5% acetic acid (∼ pH 2.0) was high (≥30 mV) at both 1 and 6 mg/mL. These values are consistent with the high abundance of His residues (and the absence of other charged residues) in suckerin-39, which are protonated under acidic conditions. We also observed that the zeta-potential of suckerin-39 in water was similar to that in 5% acetic acid at different concentrations (Table 2). The high zeta potential suggests a high colloidal stability of suckerin-39 particles, which was supported by the observation that the particle size distribution remained nearly constant for 2 weeks at 4 °C in both low and high concentrations in both solvents (Figure 4d). In addition, the zeta-potential of suckerin-39 in 0.05 M MES was 19.7 ± 0.4 mV at 0.64 mg/mL, indicating relatively stable colloidal particles of suckerin-39 as well. In summary, our biophysical investigations indicated that suckerin-39 formed soluble oligomers in 5% acetic acid, water, and MES, which can be stabilized into larger, soluble, β-sheetenriched colloids at higher concentration. While much remains to be discovered regarding the self-assembly of native SRT, we suggest that the tendency of full-length recombinant suckerins to assemble into distinct oligomeric complexes may reflect the native self-assembly behavior of these proteins, where they are likely concentrated within the sucker’s specialized epithelial cells and exported into the confined extracellular interface at the base of the SRT during its growth and development. We also suggest that soluble, β-sheet enriched nanoscale suckerinbased oligomers/colloids represent useful precursors that limit

unwanted aggregation during processing and which will facilitate the assembly and engineering of solid-state macroscopic materials that mimic the β-sheet reinforced native SRT. While synthetic silk proteins have been researched and developed over the past two decades, suckerins are newly discovered proteins. However, the suckerins exhibit several parallels with silk proteins, suggesting that they may be suitable for a range of applications. Table 3 compares and contrasts several key aspects of suckerins with silk proteins. Notably, suckerin-39 exhibits a clear ease of expression of full length proteins in E. coli and favorable solubility in aqueous solvents. Given that suckerins exhibit biocompatibility in cell culture and that they can be readily processed into a variety of structures at range of length scales,15 we suggest that they represent a new class of proteins with potential for the efficient engineering of robust β-sheet reinforced materials.

■

CONCLUSION Proteins constituting the building blocks of squid SRT display a unique combination of biophysical and mechanical properties, and their molecular structure is reminiscent to that of silks. In this study, two forms of the most abundant protein of SRT (suckerin-39) were successfully expressed in E. coli and purified by various methods. Critically, full-length suckerins were readily expressed. His-tagged suckerin-39 (His-suckerin-39) was purified from inclusion bodies by IMAC. This version of suckerin-39 may be suitable for a variety of applications. For instance the His-tag may be used for surface functionalization or metal coordination. However, in other applications the Histag must be removed, which requires expression of TEV protease followed by cleavage and final separation of TEV protease from detagged His-suckerin-39. Our data indicates that nontagged suckerin-39 is best purified using facile nonchromatographic methods including inclusion body purification by microfluidization. The achieved yields are promising, although they do not yet match those recently obtained with some silk mimics. However, we suggest that yields could be significantly optimized, by varying the bioprocessing conditions and by exploring alternative codon-optimized host expression systems. After purification, suckerin-39 exhibits in-solution characteristics that are of direct use for the for the aqueousbased, eco-friendly processing of suckerin-based biomaterials. This includes a high solubility in mild acidic conditions, which can be switched into nanometer-scale colloids stabilized by interchain β-sheet interactions upon an increase in concen3287

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

(9) Miserez, A.; Schneberk, T.; Sun, C.; Zok, F. W.; Waite, J. H. The transition from stiff to compliant materials in squid beaks. Science 2008, 319 (5871), 1816−9. (10) Miserez, A.; Rubin, D.; Waite, J. H. Cross-linking chemistry of squid beak. J. Biol. Chem. 2010, 285 (49), 38115−24. (11) Broomell, C. C.; Khan, R. K.; Moses, D. N.; Miserez, A.; Pontin, M. G.; Stucky, G. D.; Zok, F. W.; Waite, J. H. Mineral minimization in nature’s alternative teeth. J. R. Soc., Interface 2007, 4 (12), 19−31. (12) Amini, S.; Miserez, A. Wear and abrasion resistance selection maps of biological materials. Acta Biomater. 2013, 9 (8), 7895−907. (13) Guerette, P. A.; Hoon, S.; Ding, D.; Amini, S.; Masic, A.; Ravi, V.; Venkatesh, B.; Weaver, J. C.; Miserez, A. Nanoconfined β-sheets mechanically reinforce the supra-biomolecular network of robust squid sucker ring teeth. ACS Nano 2014, DOI: 10.1021/nn502149u. (14) Zok, F. W.; Miserez, A. Property maps for abrasion resistance of materials. Acta Mater. 2007, 55 (18), 6365−6371. (15) Guerette, P. A.; Hoon, S.; Seow, Y.; Raida, M.; Masic, A.; Wong, F. T.; Ho, V. H.; Kong, K. W.; Demirel, M. C.; Pena-Francesch, A.; Amini, S.; Tay, G. Z.; Ding, D.; Miserez, A. Accelerating the design of biomimetic materials by integrating RNA-seq with proteomics and materials science. Nat. Biotechnol. 2013, 31 (10), 908−15. (16) Guerette, P. A.; Ginzinger, D. G.; Weber, B. H.; Gosline, J. M. Silk properties determined by gland-specific expression of a spider fibroin gene family. Science 1996, 272 (5258), 112−5. (17) Gatesy, J.; Hayashi, C.; Motriuk, D.; Woods, J.; Lewis, R. Extreme diversity, conservation, and convergence of spider silk fibroin sequences. Science 2001, 291 (5513), 2603−5. (18) Keten, S.; Xu, Z.; Ihle, B.; Buehler, M. J. Nanoconfinement controls stiffness, strength and mechanical toughness of β-sheet crystals in silk. Nat. Mater. 2010, 9 (4), 359−67. (19) Omenetto, F.; Kaplan, D. From silk cocoon medical miracle. Scientists are crafting arteries, ligaments, circuitry and holograms from worm yarn. Sci. Am. 2010, 303 (5), 76−7. (20) Wenk, E.; Merkle, H. P.; Meinel, L. Silk fibroin as a vehicle for drug delivery applications. J. Controlled Release 2011, 150 (2), 128−41. (21) Omenetto, F. G.; KapLan, D. L. A new route for silk. Nat. Photonics 2008, 2 (11), 641−643. (22) Cappello, J.; Crissman, J.; Dorman, M.; Mikolajczak, M.; Textor, G.; Marquet, M.; Ferrari, F. Genetic-engineering of structural protein polymers. Biotechnol. Prog. 1990, 6 (3), 198−202. (23) Huang, J.; Foo, C. W. P.; Kaplan, D. L. Biosynthesis and applications of silk-like and collagen-like proteins. Polym. Rev. 2007, 47 (1), 29−62. (24) Murphy, A. R.; Kaplan, D. L. Biomedical applications of chemically-modified silk fibroin. J. Mater. Chem. 2009, 19 (36), 6443− 6450. (25) Fahnestock, S. R.; Yao, Z.; Bedzyk, L. A. Microbial production of spider silk proteins. J. Biotechnol. 2000, 74 (2), 105−19. (26) Prince, J. T.; Mcgrath, K. P.; Digirolamo, C. M.; Kaplan, D. L. Construction, cloning, and expression of synthetic genes encoding spider dragline silk. Biochemistry 1995, 34 (34), 10879−10885. (27) Lewis, R. V.; Hinman, M.; Kothakota, S.; Fournier, M. J. Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins. Protein Expression Purif. 1996, 7 (4), 400−6. (28) Fahnestock, S. R.; Irwin, S. L. Synthetic spider dragline silk proteins and their production in Escherichia coli. Appl. Microbiol. Biotechnol. 1997, 47 (1), 23−32. (29) Arcidiacono, S.; Mello, C.; Kaplan, D.; Cheley, S.; Bayley, H. Purification and characterization of recombinant spider silk expressed in Escherichia coli. Appl. Microbiol. Biotechnol. 1998, 49 (1), 31−8. (30) Tokareva, O.; Michalczechen-Lacerda, V. A.; Rech, E. L.; Kaplan, D. L. Recombinant DNA production of spider silk proteins. Microb. Biotechnol. 2013, 6 (6), 651−63. (31) Heidebrecht, A.; Scheibel, T. Recombinant production of spider silk proteins. Adv. Appl. Microbiol. 2013, 82, 115−53. (32) Lewis, R. V. Spider silk: ancient ideas for new biomaterials. Chem. Rev. 2006, 106 (9), 3762−74.

tration. This study thus suggests that recombinant suckerin proteins maintain potential for the industrial fabrication of functional submicron structures similar to the microspheres and colloids recently developed for silks63 and for the creation of βsheet reinforced materials that may find use in a range of biomedical and engineering applications.

■

ASSOCIATED CONTENT

* Supporting Information S

Tables on buffer screen results for TEV protease cleavage of His-tag of His-suckerin-39 and on solubility testing of suckerin39 in buffered saline solutions. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions †

These authors contributed equally to this manuscript (D.D. and P.A.G.). Notes

The authors declare the following competing financial interest(s): Conflict of Interest Disclosure Statement: The Authors Guerette, Hoon, and Miserez have filed a U.S. Patent on suckerin genes/proteins and their use.

■

ACKNOWLEDGMENTS We thank Dr. Luigi Petrone for assistance with FTIR data analysis and Dr. Vitali Lipik for assistance with zeta-potential measurements. We also thank the anonymous reviewers for their helpful suggestions. This research was funded by a Singapore Ministry of Education Tier 2 Grant (MOE 2011-T22-044; A.M. and J.L.), the Singapore National Research Foundation (NRF) through a NRF Fellowship (A.M.), and the Biomedical Research Council (BMRC) of the Agency for Science, Technology, and Research (A*STAR) of Singapore (S.H.).

■

REFERENCES

(1) Meyers, M. A.; Chen, P.-Y.; Lin, A. Y.-M.; Seki, Y. Biological materials: structure and mechanical properties. Prog. Mater. Sci. 2008, 53 (1), 1−206. (2) Miserez, A.; Weaver, J. C.; Pedersen, P. B.; Schneeberk, T.; Hanlon, R. T.; Kisailus, D.; Birkedal, H. Microstructural and biochemical characterization of the nanoporous sucker rings from Dosidicus gigas. Adv. Mater. 2009, 21 (4), 401−406. (3) Fratzl, P. Biomimetic materials research: what can we really learn from nature’s structural materials? J. R. Soc., Interface 2007, 4 (15), 637−42. (4) Gomes, S.; Leonor, I. B.; Mano, J. F.; Reis, R. L.; Kaplan, D. L. Natural and genetically engineered proteins for tissue engineering. Prog. Polym. Sci. 2012, 37 (1), 1−17. (5) Messenger, R. T. H. J. B. Cephalopod Behaviour;Cambridge University Press: New York, 1996. (6) Chiao, C. C.; Wickiser, J. K.; Allen, J. J.; Genter, B.; Hanlon, R. T. Hyperspectral imaging of cuttlefish camouflage indicates good color match in the eyes of fish predators. Proc. Natl. Acad. Sci. U.S.A. 2011, 108 (22), 9148−53. (7) Kier, W.; Leeuwen, J. A kinematic analysis of tentacle extension in the squid Loligo pealei. J. Exp.Biol. 1997, 200 (Pt 1), 41−53. (8) Miserez, A.; Li, Y.; Waite, J. H.; Zok, F. Jumbo squid beaks: inspiration for design of robust organic composites. Acta Biomater. 2007, 3 (1), 139−49. 3288

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289

Biomacromolecules

Article

(33) Xia, X. X.; Qian, Z. G.; Ki, C. S.; Park, Y. H.; Kaplan, D. L.; Lee, S. Y. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Natl. Acad. Sci. U.S.A. 2010, 107 (32), 14059−63. (34) Omenetto, F. G.; Kaplan, D. L. New opportunities for an ancient material. Science 2010, 329 (5991), 528−31. (35) Rockwood, D. N.; Preda, R. C.; Yucel, T.; Wang, X.; Lovett, M. L.; Kaplan, D. L. Materials fabrication from Bombyx mori silk fibroin. Nat. Protoc. 2011, 6 (10), 1612−31. (36) Hu, X.; Shmelev, K.; Sun, L.; Gil, E. S.; Park, S. H.; Cebe, P.; Kaplan, D. L. Regulation of silk material structure by temperaturecontrolled water vapor annealing. Biomacromolecules 2011, 12 (5), 1686−96. (37) van den Berg, S.; Lofdahl, P. A.; Hard, T.; Berglund, H. Improved solubility of TEV protease by directed evolution. J. Biotechnol. 2006, 121 (3), 291−8. (38) Asial, I.; Cheng, Y. X.; Engman, H.; Dollhopf, M.; Wu, B.; Nordlund, P.; Cornvik, T. Engineering protein thermostability using a generic activity-independent biophysical screen inside the cell. Nat. Commun. 2013, 4, 2901. (39) Waugh, D. S. An overview of enzymatic reagents for the removal of affinity tags. Protein Expression Purif. 2011, 80 (2), 283−93. (40) Arnau, J.; Lauritzen, C.; Petersen, G. E.; Pedersen, J. Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expression Purif. 2006, 48 (1), 1−13. (41) Joseph, E. Tropea, S. C., and Waugh, D. S. Expression and Purification of Soluble His6-Tagged TEV Protease. In Methods in Molecular Biology: High Throughput Protein Expression and Purification; Doyle, S. A., Ed.; Humana Press: New York, 2009; p 297. (42) Sun, C.; Liang, J.; Shi, R.; Gao, X.; Zhang, R.; Hong, F.; Yuan, Q.; Wang, S. Tobacco etch virus protease retains its activity in various buffers and in the presence of diverse additives. Protein Expression Purif. 2012, 82 (1), 226−31. (43) Fahnestock, S. R.; Bedzyk, L. A. Production of synthetic spider dragline silk protein in Pichia pastoris. Appl. Microbiol. Biotechnol. 1997, 47 (1), 33−9. (44) Zhang, Y.; Hu, J.; Miao, Y.; Zhao, A.; Zhao, T.; Wu, D.; Liang, L.; Miikura, A.; Shiomi, K.; Kajiura, Z.; Nakagaki, M. Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield. Mol. Biol. Rep 2008, 35 (3), 329−35. (45) Scheller, J.; Guhrs, K. H.; Grosse, F.; Conrad, U. Production of spider silk proteins in tobacco and potato. Nat. Biotechnol. 2001, 19 (6), 573−7. (46) Lazaris, A.; Arcidiacono, S.; Huang, Y.; Zhou, J. F.; Duguay, F.; Chretien, N.; Welsh, E. A.; Soares, J. W.; Karatzas, C. N. Spider silk fibers spun from soluble recombinant silk produced in mammalian cells. Science 2002, 295 (5554), 472−6. (47) Collins, T.; Barroca, M.; Branca, F.; Padrao, J.; Machado, R.; Casal, M., High level biosynthesis of a silk-elastin-like protein in E. coli. Biomacromolecules 2014. (48) Teng, W.; Cappello, J.; Wu, X. Recombinant silk-elastinlike protein polymer displays elasticity comparable to elastin. Biomacromolecules 2009, 10 (11), 3028−36. (49) Gustafson, J.; Greish, K.; Frandsen, J.; Cappello, J.; Ghandehari, H. Silk-elastin-like recombinant polymers for gene therapy of head and neck cancer: from molecular definition to controlled gene expression. J. Controlled Release 2009, 140 (3), 256−61. (50) Xia, X. X.; Xu, Q.; Hu, X.; Qin, G.; Kaplan, D. L. Tunable selfassembly of genetically engineered silk-elastin-like protein polymers. Biomacromolecules 2011, 12 (11), 3844−50. (51) Elvin, C. M.; Carr, A. G.; Huson, M. G.; Maxwell, J. M.; Pearson, R. D.; Vuocolo, T.; Liyou, N. E.; Wong, D. C.; Merritt, D. J.; Dixon, N. E. Synthesis and properties of crosslinked recombinant proresilin. Nature 2005, 437 (7061), 999−1002. (52) Greenfield, N. J. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 2006, 1 (6), 2876−90.

(53) Lu, Q.; Hu, X.; Wang, X.; Kluge, J. A.; Lu, S.; Cebe, P.; Kaplan, D. L. Water-insoluble silk films with silk I structure. Acta Biomater. 2010, 6 (4), 1380−7. (54) Fen, N.; DeOliveira, D. B.; Trumble, W. R.; Sarkar, H. K.; Singh, B. R. Secondary structure estimation of proteins using the amide III region of Fourier transform infrared spectroscopy: application to analyze calcium-binding-induced structural changes in calsequestrin. Appl. Spectrosc. 1994, 48 (11), 1432−1441. (55) Barth, A.; Zscherp, C. What vibrations tell us about proteins. Q. Rev. Biophys. 2002, 35 (4), 369−430. (56) Kong, J.; Yu, S. Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochim. Biophys. Sin. 2007, 39 (8), 549−59. (57) Gosline, J. M.; Guerette, P. A.; Ortlepp, C. S.; Savage, K. N. The mechanical design of spider silks: from fibroin sequence to mechanical function. J. Exp. Biol. 1999, 202 (Pt 23), 3295−303. (58) Zhang, K.; Si, F. W.; Duan, H. L.; Wang, J. Microstructures and mechanical properties of silks of silkworm and honeybee. Acta Biomater. 2010, 6 (6), 2165−71. (59) Ebenstein, D. M.; Wahl, K. J. Anisotropic nanomechanical properties of Nephila clavipes dragline silk. J. Mater. Res. 2006, 21 (08), 2035−2044. (60) Sponner, A.; Schlott, B.; Vollrath, F.; Unger, E.; Grosse, F.; Weisshart, K. Characterization of the protein components of Nephila clavipes dragline silk. Biochemistry 2005, 44 (12), 4727−36. (61) Ayoub, N. A.; Garb, J. E.; Tinghitella, R. M.; Collin, M. A.; Hayashi, C. Y. Blueprint for a high-performance biomaterial: fulllength spider dragline silk genes. PLoS One 2007, 2 (6), e514. (62) Vepari, C.; Kaplan, D. L. Silk as a biomaterial. Prog. Polym. Sci. 2007, 32 (8−9), 991−1007. (63) Lammel, A. S.; Hu, X.; Park, S. H.; Kaplan, D. L.; Scheibel, T. R. Controlling silk fibroin particle features for drug delivery. Biomaterials 2010, 31 (16), 4583−91.

3289

dx.doi.org/10.1021/bm500670r | Biomacromolecules 2014, 15, 3278−3289