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Recombinant production and film properties of full-length hornet silk proteins
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Recombinant production and film properties of full-length hornet silk proteins
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Yusuke Kambe a, Tara D. Sutherland b, Tsunenori Kameda a,⇑ a b
Silk Materials Research Unit, National Institute of Agrobiological Sciences (NIAS), 1–2 Owashi, Tsukuba, Ibaraki 305-8634, Japan Ecosystem Sciences, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clunies Ross St., Acton, ACT 2601, Australia
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 1 May 2014 Accepted 15 May 2014 Available online xxxx Keywords: Biomimetic material Fourier transform infrared spectroscopy Mechanical test Surface analysis Isoelectric point
a b s t r a c t Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1–4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160–330 mg recombinant protein l 1. Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (f) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1–4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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Silks are fibrous protein materials produced by a large number of arthropods [1]. The silk of the domesticated silkworm Bombyx mori has been used in medicine for centuries as surgical suture and, more recently, degummed silk has been FDA-approved for clinical use [2]. The native silk proteins have been reconstituted from natural silk and processed into films, meshes, gels and sponges for applications such as scaffolds for tissue engineering [3–5]. Besides their mechanical properties, these materials possess other characteristics essential for scaffolds: they are biodegradable and non-cytotoxic, and they generate low-immunity and low-inflammatory reactivities [3,6–8]. However, silk scaffolds reportedly show low/weak cell adhesion [9–12], because most of the silk proteins have no cell adhesive amino acid sequences. Additionally, transgenic technologies, aimed at producing a homogeneous supply of the silk proteins and enabling the modification of the proteins towards specific biocompatibility and functional requirements, have been unsuccessful in generating commercial scale processes. The expression efficiency of proteins in recombinant B. mori silkworms remains low (at most 15%) [13], and the large (>10 kbp) and repetitive silk gene sequences
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⇑ Corresponding author. Tel./fax: +81 29 838 6213. E-mail address: [email protected] (T. Kameda).
have limited other recombinant systems to the expression of fragments of the silk proteins [14,15]. In addition to the well-known cocoons produced by silkworms and webs produced by spiders, social hymenopterans, including honeybees, ants and hornets, also produce ample amounts of silk. The principal molecular structure of the social hymenopteran silk is a-helices, frequently in a coiled-coil conformation [16], a molecular structure distinct from the b-crystalline structures that dominate the silks of silkworms and spiders. Full-length silk genes from a number of social hymenopteran species have been identified and cloned [15,17,18]. The small size (1 kbp) and comparatively low levels of repetition in these genes have allowed recombinant production of full-length honeybee silk proteins, AmelF1–4, in E. coli at high yield and potentially on a large scale [19]. Silk cocoons produced by larvae of yellow hornets, Vespa simillima (Fig. 1), consist of four major proteins: Vssilk1–4 [18,20]. Vssilk1–4 are homologous to AmelF1–4 and are also small (30–70 kDa) and non-repetitive, in contrast to silk proteins of silkworms and spiders. Unlike ant and honeybee silks, V. simillima silk (hornet silk) can be dissolved in concentrated salt solutions (e.g. 9 M LiBr) [20,21], probably owing to the lower presence of e-(cglutamyl)-lysine crosslinks [21]. The solubility of the hornet silk in salt solutions would lead its reconstitution into highly pure products, because the salt can be removed by simple methods such as dialysis. Reconstituted hornet silk can be readily processed into
http://dx.doi.org/10.1016/j.actbio.2014.05.013 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kambe Y et al. Recombinant production and film properties of full-length hornet silk proteins. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.05.013
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Fig. 1. Photograph of silk cocoons of yellow hornets (Vespa simillima).
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transparent films that are flexible and with mechanical properties suitable for surgical handling [22]. The mechanical properties of these hornet silk films include a breaking tensile strain of 100%, a maximum tensile strength of 170 MPa and tensile modulus of 5.5 GPa [23], values superior to those of films fabricated from reconstituted B. mori silk proteins [24,25]. As found in other silks, hornet silk is dominated by the small amino acids Ala and Ser (35% and 23%, respectively). However, the four hornet silk proteins also contain significant levels (16%) of charged amino acids (Arg, Asp, Glu and Lys). The charged amino acid composition of each of the proteins is different, resulting in unique predictions for the isoelectric point (pI) for each protein (ranging from 4.4 to 10.3 [18]). In contrast, the predicted pI of the four honeybee silk proteins, AmelF1–4, are similar (from 5.1 to 5.8 [18]). B. mori silk fibroin, which is composed of a heavy chain (H-chain) and a light chain (L-chain), has a pI of around five [8]. Hence, the hornet silk is composed of a complex assembly of multi-silk proteins, each with different physicochemical properties. These findings suggest that the hornet silk can be expected to be fabricated into highly pure scaffolds that provide specific physical and chemical environments for various types of cells, depending on varied combinations of the four Vssilk proteins. However, the homologous preparation of each of the four silk proteins has not been established, and their characteristics remain unclear. The goal in the present study was to produce full length versions of the four Vssilk proteins in E. coli in order to investigate the structure, mechanical performance and physicochemical surface properties of materials generated from the individual proteins. Cast films from each Vssilk protein were generated and compared with films generated from reconstituted native hornet silk and reconstituted B. mori silk. The results indicated that films generated from recombinant Vssilk proteins have specific surface wettability and charge properties conducive to cell adhesion to materials.
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2. Materials and methods
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2.1. Recombinant production of four hornet silk proteins
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To prepare vectors for the expression of Vssilk1–4, the four hornet silk gene sequences (accession no. AB537885, AB537886, AB537887 and AB537888) without signal peptides were amplified by polymerase chain reaction (PCR) from the previously reported cDNA clones [18]. The four silk proteins have similar tri-block architecture, and the full amino acid sequence of the expressed proteins is shown in Supplementary Data Fig. 1. The codon for the first amino acid (Ser) of the native Vssilk2 protein was replaced by a codon for Gly to enable cloning into the vector NcoI site. The
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oligonucleotide primers used to amplify cDNA prior to cloning into expression vectors were as follows, with NcoI, NdeI and BamHI sites underlined and sequences that match the cDNA italicized: Vssilk1: GGATTCCC ATG GGG CCA TCA AGG TTG TCT GAA ACC and CGGCCATATG TTA TTA GGC GCT GCT ACT ACT CGA AGA GGC; Vssilk2: GGATTCCC ATG GCG GCA GCG ATC GCA TCG GCT CGA and CGGCGGATCC TTA TTA GTT TCC GGA TGC GCT GCG AGA ACT; Vssilk3: GGATTCCC ATG GCA GAA AGC TCA AGC TCA AGC TCC and CGGCGGATCC TTA TTA CCA ATC CTG GGA CCA GAT ATT CTG; Vssilk4: GGATTCCC ATG GAC AGA TCG TGG GCA GCA TCG GAT and CGGCGGATCC TTA TTA GTT TAA GAT CAG CGA GCT AGT TCT. The PCR products were digested with appropriate restriction enzymes (Vssilk1: NcoI and NdeI; and Vsilk2–4: NcoI and BamHI; New England Biolabs) and cloned into the pET14b expression vector (Novagen) using T4 DNA ligase (New England Biolabs). The constructs were verified by restriction digest and DNA sequencing before expression. E. coli strain Rosetta 2 (DE3) competent cells (Novagen), transformed with the vector DNA, were plated onto LB agar containing 150 lg ml 1 ampicillin and 50 lg ml 1 chloramphenicol. After overnight incubation at 37 °C, recombinant colonies were transferred to Overnight Express Instant TB medium (Novagen) with 10% (v/v) glycerol in shake flasks and then incubated for 48 h at 25 °C or 24 h at 28 or 31 °C. Cultures were pelleted by centrifugation at 10,000g for 10 min at 4 °C and stored at 20 °C until use.
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2.2. Analysis of expression levels of recombinant hornet silk proteins
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The soluble fraction and inclusion bodies from recombinant E. coli were separated using BugBuster Master Mix (Novagen) according to the manufacturer’s protocol. Inclusion bodies were further purified after the cells were resuspended in five times their volume of the BugBuster reagent and incubated for 30 min at room temperature (RT). After centrifugation at 10,000g for 10 min at 4 °C, the pellet was washed with five times its volume of the BugBuster reagent, followed by washing in five times its volume of the BugBuster reagent diluted tenfold with phosphate-buffered saline (PBS). The purified inclusion bodies were resuspended in 9 M LiBr aqueous solution, incubated at 70 °C for 30 min and centrifuged at 10,000g for 10 min at 4 °C to remove bacterial proteins. The solutions of essentially pure Vssilk1, Vssilk2, Vssilk3 or Vssilk4 in 9 M LiBr were filtered through a 0.22-lm membrane (Merck) to remove insoluble matter. The solutions were then diluted with four times volume of reverse osmosis (RO) water, enough not to affect the following concentration analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [20]. BCA protein assay kits (Pierce) were used to determine the concentration of the protein and to calculate expression levels. The protein composition of the Vssilk1–4/LiBr solutions was analyzed by SDS-PAGE on Mini-PROTEAN TGX gels (Any kD; Bio-Rad) under a reducing condition with the intensity of protein bands measured by ImageJ (NIH).
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2.3. Films of native hornet silk, recombinant Vssilk 1–4 and silk fibroin
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Cocoons of V. simillima were collected from their nest and dissolved in 9 M LiBr solution by stirring for 1 h at 37 °C, followed by filtering through a 0.22-lm membrane to remove the nest papers adhering to the cocoons. B. mori silk/LiBr solution was prepared by dissolving degummed fibers of cocoons in 9 M LiBr solution at RT overnight. The recombinant Vssilk1–4, native hornet silk and B. mori silk / 9 M LiBr solutions were dialyzed against RO water, using cellulose dialysis membranes (MWCO, 6000–8000; Spectrum Laboratories) for 3 days at 4 °C, changing the water every 10–14 h, resulting in
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the almost complete removal of Li+ and Br ions [26]. During the dialysis, the solutions, except the silk fibroin solution, tuned into a turbid gel or resulted in a precipitate. The silk gels, precipitates or aqueous solutions were freeze-dried. The resultant dried silk proteins were immediately weighed and dissolved in hexafluoroisopropyl alcohol (HFIP) by stirring at RT overnight to prepare 1% (w/v) silk protein/HFIP solutions. Films of recombinant Vssilk1, Vssilk2, Vssilk3 and Vssilk4, native hornet silk and silkworm silk fibroin were prepared by spreading the protein/HFIP solution over the following plates and dishes at 100 ll cm 2: borosilicate glass plates (24 60 mm; Matsunami Glass) for structural and surface analyses; 50-mm polystyrene dishes (Eiken Chemical) for tensile testing; and 24well polystyrene plates (IWAKI) for cell culture. After spreading the solution, the plates and dishes were air dried at RT for 24 h. Films on the plates and dishes were then immersed in 80% (v/v) methanol at RT for 1 h, to make them water insensitive, and air dried at RT for 24 h. Following this, the films were sterilized by submerging in 70% (w/w) ethanol for 20 min, rinsed twice with autoclaved ultrapure water, and air dried at RT for 24 h. The resultant silk films were photographed under RT air conditions using a digital camera (EOS-10D; Canon).
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2.4. Fourier transform infrared spectroscopy (FTIR)
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FTIR analysis of the silk films (as-cast, after treatment with 80% (v/v) methanol, and after sterilization with 70% (w/w) ethanol) was performed using a spectrometer (FT/IR-620 and IRT-30; Jasco), equipped with a single-reflection Ge ATR attachment (ATR-30-G45; Jasco). The instrument was continuously purged with nitrogen gas to remove atmospheric water vapor. The spectra were recorded with an accumulation of 128 scans at a resolution of 4 cm 1.
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2.5. Mechanical testing of silk films
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For tensile testing of the silks, the films were gently peeled from the polystyrene dishes and cut into strips 2.0 mm wide and 30.0 mm long. The thickness of the films was estimated to be 4.9 ± 1.0 lm by electron microscopy (TM-1000; Hitachi). The films were stored in a desiccators until use. The tensile properties of the dry specimens were measured using a tensile tester (EZ test; Shimadzu), equipped with a 5 N load cell, at a rate of 5 mm min 1. The gauge length was set at 20 mm. Tests were conducted in air at 24–26 °C and 41–48% relative humidity. From the stress/strain curves, maximum stress, breaking stress, breaking strain and toughness were determined. The tensile moduli were determined from the initial slope of the stress/strain curves at a strain of 1– 1.2%. Nine different samples were tested (n = 9).
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2.6. Measurement of surface properties of silk films
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Static water contact angles on the films were determined by the sessile drop method, using a contact angle analyzer (FTA188; First Ten Angstroms) at RT. A droplet (3.5 ll) of ultrapure water was placed on the film surface, and the time-dependent changes of the contact angle were recorded for 90 s. The water contact angle was determined as the intercept contact angle (at 0 s) of a line fitted between 30 and 60 s. Eight different areas were analyzed (n = 8). The zeta (f) potential of each film was measured using a fpotential analyzer (ELS-8000; Otsuka Electronics), equipped with the cell unit for flat plate samples, in 5 mM sodium phosphate buffer and 5 mM NaCl at 25 °C. The pH of the buffer was adjusted to 3.00, 4.00, 5.00, 6.00, 7.00, 8.00 and 9.00 with HCl and NaOH aqueous solutions. A sample was used to measure f potential at the seven pH by changing the streaming solution. Three different
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samples were used (n = 3). Curve fitting was done using two- or three-degree polynomial regression for each sample, and the f potential at pH 7.4 and pI of the sample were calculated.
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2.7. Cell culture on silk films
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The ability of the silken films to promote cell attachment was measured after NIH3T3 murine fibroblasts in Eagle’s minimum essential medium (Nissui Parmaceutical) supplemented with 10% (v/v) fetal bovine serum (Gibco), 10 ng ml 1 kanamycin (Gibco), 0.2% sodium bicarbonate (Gibco) and 2 mM l-Glu (Gibco) were seeded onto the films prepared in 24-well cell culture plates at 5.0 103 cells cm 2. The films and cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for up to 24 h, and cell morphology and distribution on the films were observed using a phase-contrast inverted microscope (IX-70, Olympus), equipped with a cooled CCD camera (TCH5.0ICE; Xintu Photonics). The number of cells that had adhered to the silk films was determined using the previously described lactate dehydrogenase (LDH) assay [27]. Briefly, after incubation, the cells were washed twice with PBS. Washed cells were then dissolved by overnight incubation in 0.5% (w/v) Triton X-100 (Research Organics) in PBS at 4 °C. The LDH activity of the dissolved cells was measured from changes in the absorbance at 340 nm that are attributed to the kinetics of nicotinamide adenine dinucleotide-consuming reactions. The adherence of fibroblasts cultured on tissue culture polystyrene (TCPS; IWAKI) were measured as a positive control. Four different films were used for each sample point (n = 4).
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2.8. Statistical analysis
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The tensile and surface properties (water contact angle, f potential at pH 7.4, and pI) of the various films were compared using one-way ANOVA, followed by Dunnett post hoc analysis, using the measurements from the native hornet silk films as the control. The effects of the type of film and culture time on cell number were analyzed using two-way ANOVA, followed by Dunnett post hoc comparisons with the native hornet silk film as the control. A value of P < 0.05 was considered significant. In all figures, asterisks indicate statistical differences from the native hornet silk film.
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3. Results
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3.1. Production of recombinant hornet silk proteins and analysis of film impurities
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The four full-length hornet silk proteins (Vssilk1–4) were successfully synthesized after expression from transgenic E. coli in shake flasks. At all culture temperatures investigated in this study (25, 28, and 31 °C), SDS-PAGE (data not shown) indicated that the silk proteins were more highly expressed in inclusion bodies than in the soluble form. Highest levels of expression were achieved after 48 h expression at 25 °C (Vssilk1, 3 and 4), and after 24 h expression at 31 °C for Vssilk2. Proteins appeared not to be solubilized from the inclusion bodies with SDS, a detergent commonly used in molecular biology to analyze proteins, and therefore only slight amount of the proteins were observed on SDS-PAGE gels without further treatment. The inclusion bodies could be solubilized in 9 M LiBr, an ionic solution used to reconstitute native hornet and silkworm silk. Under these conditions, 330, 170, 160 and 330 mg culture medium of Vssilk1, 2, 3, and 4 proteins, respectively, were achieved at a high purity (>90%; Fig. 2). After removal of LiBr and processing into films, as described in Section 2, solid-state 13C CP/MAS nuclear magnetic resonance (NMR) analysis of recombinant hornet silk films generated from these proteins (Supplementary Data Fig. 2) showed that
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Fig. 2. SDS-PAGE of recombinant hornet silk proteins Vssilk1–4. Lane numbers correspond to recombinant V. simillima hornet silk proteins Vssilk1–4. N indicates the lane for native hornet silk proteins. NovexÒ Sharp Protein Standard (Invitrogen) was used as molecular weight markers (M).
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no BugBuster detergent, LiBr or HFIP derived impurities were contained in the final recombinant films. As shown in Fig. 3, the silk films were generally smooth and transparent, with the Vssilk1 film slightly opaque.
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3.2. Molecular structure of proteins in silk film
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The secondary structures of the proteins in the various recombinant silk films were compared with those in films generated from
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native hornet and silkworm silk using FTIR (Fig. 4). In FTIR spectra, the signal at 1650 and 1624 cm 1 can be attributed to a-helix and b-sheet conformations, respectively. Initially, all the silk films contained a predominantly a-helix structure (Fig. 4a). Submersion in an 80% (v/v) methanol and drying (Fig. 4b) induced a conformational transition from the a helix to the b sheet, particularly in the B. mori silk film. In contrast, the native hornet silk and recombinant Vssilk films consisted of coexisting a-helix and b-sheet conformations, with the Vssilk4 film being comparatively rich in b-sheets in comparison with the other hornet silk films. Immersion in 70% (w/w) ethanol and drying (Fig. 4c), which served to sterilize the films, promoted further transition to b-sheet structure in all films. However, all hornet silk films maintained both a-helix and b-sheet structures. In comparison with the native hornet silk film, the recombinant Vssilk1, 2 and 3 films were rich in a-helices, whereas the Vssilk4 film was rich in b-sheet structure.
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3.3. Tensile properties of silk films
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The mechanical properties of the sterilized silk films in dry conditions are shown in Table 1. All the recombinant Vssilk films showed mechanical properties comparable with or superior to the native hornet silk film, with the exception of the breaking strain of the Vssilk2 film. In particular, the films of Vssilk3 or 4 proteins were more ductile than the native hornet silk film, without a decrease in tensile modulus. The B. mori silk film was harder and brittler than the hornet silk films, showing the lowest breaking strain and toughness, but the highest tensile modulus among the films tested in this study.
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3.4. Physicochemical properties of silk films
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The water contact angles on the various silk films are shown in Table 2. The contact angle measurements for the Vssilk1 and 2 films are significantly higher than those for the native hornet silk film, indicating that the surface of the Vssilk1 or 2 films are more hydrophobic. There are no significant differences between either the Vssilk3 or Vssilk4 and native hornet silk films. The B. mori silk film had a more hydrophilic surface than any of the hornet silk films. Fig. 5 shows the f potential of the silk films in different pH environments. The profile of f potential across different pH
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Fig. 3. Photographs of HFIP cast silk films after treatments with 80% (v/v) methanol and 70% (w/w) ethanol followed by washing with ultrapure water. Scale bar = 2 cm.
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Fig. 4. FTIR spectra of the amide I region of films of recombinant hornet silk and reconstituted native hornet and silkworm silks. The signal at 1650 and 1624 cm 1 are attributed to the a-helix and b-sheet conformations, respectively. In all figures, dotted lines indicate the spectrum for native hornet silk films. (a) Cast film, (b) after treatment with 80% (v/v) methanol, and (c) after sterilization with 70% (w/w) ethanol followed by washing with ultrapure water.
Table 1 Tensile properties of silk films. Film Hornet silk
Native Vssilk1 Vssilk2 Vssilk3 Vssilk4
B. mori silk
Maximum stress (MPa)
Breaking stress (MPa)
Breaking strain (%)
Toughness (mJ)
Tensile modulus (GPa)
54 ± 6 62 ± 8 56 ± 17 62 ± 5 74 ± 17⁄⁄
45 ± 12 56 ± 9 55 ± 17 56 ± 4 73 ± 17⁄⁄⁄
3.8 ± 0.6 4.4 ± 0.5 3.1 ± 0.6⁄ 4.8 ± 0.5⁄⁄ 4.6 ± 0.7⁄
0.25 ± 0.04 0.32 ± 0.07 0.21 ± 0.12 0.38 ± 0.08⁄⁄ 0.38 ± 0.07⁄⁄
2.2 ± 0.4 2.1 ± 0.6 2.5 ± 0.7 2.3 ± 0.4 2.4 ± 0.4
57 ± 19
57 ± 19
2.3 ± 0.7⁄⁄⁄
0.13 ± 0.07⁄⁄
2.9 ± 0.6⁄
Data shown in the form: mean ± SD (n = 9). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).
Table 2 Biophysical properties of silk films. Film Hornet silk
B. mori silk
Water contact angle (deg) Native Vssilk1 Vssilk2 Vssilk3 Vssilk4
73.1 ± 4.0 84.6 ± 5.3⁄⁄⁄ 90.7 ± 5.3⁄⁄⁄ 74.3 ± 9.2 70.0 ± 5.2 63.1 ± 1.6⁄⁄
f potential at pH 7.4 (mV) 19.5 ± 6.7 +6.8 ± 1.8⁄⁄⁄ +4.5 ± 3.8⁄⁄⁄ 14.1 ± 5.1 29.9 ± 8.4 25.1 ± 1.9
Empirical pI
Calculated pI
4.7 ± 0.1 8.9 ± 0.5⁄⁄⁄ 9.1 ± 1.9⁄⁄⁄ 5.0 ± 0.1 4.2 ± 0.0 3.5 ± 0.4
ND 10.29 [14] 10.18 [14] 5.76 [14] 4.38 [14] 4.39 (H-chain) [8] 5.06 (L-chain) [8]
Data shown in the form: mean ± SD (n = 8 for water contact angle; n = 3 for f potential and pI). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001). ND: not determined.
Fig. 5. f potential in different pH environments of (A) native or recombinant hornet silk films and (B) B. mori silk film. Curve fitting was done using two- or three-degree polynomial regression. Data are shown in the form: mean ± SD (n = 3).
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Fig. 6. (A) Photographs and (B) quantitative data of fibroblast attachment to recombinant hornet silk protein, native hornet silk and native B. mori silk films after 12 and 24 h. Scale bar = 100 lm in Fig. 6A. For Fig. 6B, the vertical axis indicates the proportion of cells attached relative to the number of cells attached to TCPS dishes. Data are shown in the form: mean ± SD (n = 4). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄P < 0.05, ⁄⁄P < 0.01).
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environments of the Vssilk1 and 2 films were significantly different from those of the native hornet silk film and Vssilk3 and 4 films; the Vssilk1 and 2 films tended to show positive charges throughout the pH 3–9 range, whereas the others had pI (pH at f potential = 0) between pH 4 and 5 (Fig. 5A). Hence, the surface of Vssilk1 and 2 films had a slightly positive charge under physiological conditions (pH 7.4), whereas the native hornet silk films and Vssilk3 and 4 films were strongly negatively charged (Table 2). The B. mori silk film had pI between the pH 3 and 4 (Fig. 5B) and was also strongly negatively charged at pH 7.4 (Table 2). The empirical pI determined from regression curves corresponded approximately to pI calculated using the ProtParam tool (http://web.expasy.org/protparam/) (Table 2).
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3.5. Cell adhesion to silk films
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Cell morphology and distributions of NIH3T3 fibroblasts on the various silk films after 12 and 24 h post-seeding are shown in Fig. 6A. The surface of the silk films was non-uniform; particles (native hornet silk and Vssilk2), cracks (native hornet silk and Vssilk3) and rough surface (Vssilk1 and B. mori silk) appeared. Although all the silk films had a wavy surface, this pattern was also seen on the surface of the TCPS (data not shown). However, no tendencies of cell adhesion, such as distribution and orientation, were observed depending on these surface structures of the silk films. In contrast, cell morphologies varied, depending on the type of silk proteins. Fibroblasts tended to gather on the native hornet silk, Vssilk3 and B. mori silk films, whereas cells spread out on the films generated from Vssilk1, 2 and 4 proteins. A comparison of the number of the cells attached to the various silk films is shown in Fig. 6B. The cell number is displayed as the ratio of attached cells in comparison with the number observed attached to TCPS after the equivalent time period. The number of
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cells on the Vssilk1 or 2 films was comparable to that on TCPS and significantly larger than that on either native hornet silk films or B. mori silk films after both 12 and 24 h in culture. Cell attachment to the Vssilk3 and 4 films was not significantly different from that observed on native hornet silk films or B. mori silk films after 24 h in culture.
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4. Discussion
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In this work, recombinant full-length hornet silk proteins, Vssilk1–4, were produced in shake flasks from recombinant E. coli at yields of 160–330 mg from a liter of culture medium. Given that yield gains can be expected if expression is optimized in fermentation facilities rather than shake flasks, the yields of recombinant Vssilk rival those seen in other systems, including honeybee silk proteins at 0.2–2.5 g l 1 [19], native-sized spider silk protein at 0.5–2.7 g l 1 [28], a partial-length spider silk at 0.36 g l 1 [29] and full-length mantis fibroins at 40–400 mg l 1 [30]. The high yields of recombinant hornet silk production probably stem from the smaller size and low repetition in the sequences of silk genes. Standard analysis of proteins involves SDS-PAGE to determine protein size and purity. Previously, the honeybee silks were purified from inclusion bodies using SDS [19], the detergent promoting the folding of the silk proteins into a-helices [31]. However, it was difficult to dissolve the inclusion bodies in the present study in SDS. Saturated LiBr, a salt commonly used to reconstitute native silkworm and hornet silks, did solubilize the inclusion bodies, and the solution contained highly pure Vssilk proteins (Fig. 2). Concentrated LiBr solution completely solubilizes hornet silk [20,21], allowing insoluble impurities to be removed by centrifugation and filtration. Following removal of impurities, the silk proteins could be recovered from solution after inducing their precipitation
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by the removal of Li+ and Br ions after dialysis against RO water [23]. The silk proteins could then be recovered from the solution, freeze dried and processed into materials that did not have residual detergent or solvent present (Supplementary Data Fig. 2). It is generally known that silkworm silk proteins dissolved in HFIP solution tend to form helix conformations, and this conformation is maintained in films fabricated from these solutions [32]. The helix conformation in these silk products is transformed into b-sheet by treatment in alcohol [33–35]. The dominant molecular structure in native silkworm silk is b-sheet. Similarly, the same molecular changes in silkworm silk film were found in this study. The molecular structure of proteins in native hornet silk is different from that found in silkworm silk. The molecular architecture of the Vssilk1–4 proteins involves a central Ala-rich region that adopts an a-helical conformation, and Ser-rich regions at both ends that adopt the b-sheet conformation [18,23,36]. Bioinformatics predicts that a significant proportion of the central a-helices in the Ala-rich region forms coiled-coil structures [18,23], similar to the coiled-coil structures observed in honeybee silk. As with B. mori silk films, all the films cast from hornet proteins in HFIP solutions were rich in a-helices (Fig. 4a), and methanol and ethanol treatments promoted the formation of b-sheets (Fig. 4b and c). However, in contrast to the dramatic change in b-sheets observed in B. mori silk films, alcohol treatments induced a smaller change, and the final films maintained the coexistence of a-helices and b-sheets similar to that observed in native hornet silk films. Within the recombinant hornet silk films, the proportion of a-helix to b-sheet varied; a-helices dominated films of Vssilk1–3, whereas b-sheets dominated the Vssilk4 films (Fig. 4c). Previously, the fraction of coiled-coil structures in Vssilk1–3 was determined as 60%, but only 50% in the Vssilk4 protein [37]. The agreement between these results suggests that a-helices observed in the recombinant Vssilk films are predominantly in coiled-coil structures that are preserved after the immersion in aqueous alcohol. It is likely that the coiled-coil structure prevents transition of a-helices to b-sheets. Overall, the structural analysis indicates that the molecular structure in films generated from single recombinant Vssilk proteins mimics that in native hornet silk film with coexisting coiled-coil/a-helices and b-sheets. The recombinant Vssilk films had mechanical properties either equaling or surpassing those of the native hornet silk and B. mori silk films (Table 1). The retention of mechanical performance in films generated from a single Vssilk protein agrees with previous work by Sutherland et al. [38], which found that fibers generated from a single recombinant honeybee silk protein, AmelF3, have mechanical properties similar to fibers generated from all four of the proteins found in native honeybee silk. The finding that a single Vssilk protein can mimic the structural and mechanical properties of native hornet silk supports the conclusion that the four paralogous silk genes have been conserved for 150 million years in each social hymenopteran lineage, as a result of their protein products playing a critical role in the process of natural silk fabrication rather than in conferring properties to the end product [38]. Physicochemical surface properties of the Vssilk films varied: films from Vssilk3 and 4 possessed surface properties similar to the native hornet silk and B. mori silk fibroin, whereas the films generated from Vssilk1 and 2 were more hydrophobic and with positive surface charges (Fig. 5 and Table 2). These characteristics, particularly f potential on the silk film surface, seemed to be influenced primarily by the amino acid composition of the silk proteins, as the predicted pI of the silk proteins corresponded well to the experimental results. It is widely accepted that cell adhesion to materials is strongly influenced by the materials’ surface properties, including roughness, wettability and charge [39–48]. Since the primary structure of the Vssilk1–4 contains no specific integrin-binding motifs such as RGD, PHSRN and YIGSR (Supplemen-
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tary Data Fig. 1), the physicochemical surface properties of the silk films play an important role in promoting cell attachment (Fig. 6). Of the properties, the surface topology of the films was unlikely to heavily affect cell adhesion, because no strong tendencies were observed, depending on the topology (Fig. 6A). As for cell numbers in this study (Fig. 6B), the best cell adhesion was observed on the hydrophobic, positively charged films generated from Vssilk1 and 2 proteins. Generally, these properties have conflicting effects on cell adhesion, with other studies reporting inhibition of cell attachment to surfaces with water contact angle of >90° [41,47] and enhancement of cell adhesion to materials with positive charges [45,46]. In silken materials, silks with low pI values, i.e. negatively charged under physiological conditions, generally show weaker cell adhesion than silks with high pI values [8]. The present authors propose that the specific positive surface charges on the surface of films generated from Vssilk1 or 2 enhance cell adhesion as a result of cell–substrate interactions medicated by adsorbed negatively charged extracellular matrix proteins such as type I collagen (pI 5.46) and fibronectin (pI 5.60). Interestingly, among the recombinant hornet silk films, only fibroblasts on Vssilk3 film showed morphology similar to the cells on the native hornet silk and B. mori silk films (Fig. 6A). Although the four silk proteins in social hymenopteran silks are considered to be expressed and produced at approximately equivalent levels [49,50] to assemble into a hetero-tetrameric coiled-coil [16,36], it is inferred that Vssilk3 proteins were selectively exposed to the surface of the native hornet silk film, which resulted in similar wettability, charge and cytocompatibility of both film surfaces (Table 2 and Fig. 6). Polymer-based scaffolds can be classified into synthetic (e.g. poly(glycolic acid) and poly(lactic acid)) and natural polymers (e.g. collagen and fibrin) [51]. The synthetic polymers provide suitable bulk, flexibility and mechanical properties, but they generally lack chemical cues to stimulate cell adhesion and proliferation [51,52] and require harsh chemicals for polymerization and modification [51]. In contrast, natural polymers, particularly collagen, show biomimetic structure and cytocompatibility [51,53], but they need chemical treatments to generate suitable mechanical properties [52], and there remains a risk of immunity/infection hazards from residual antigens [53]. B. mori silk minimizes these disadvantages, but it shows only low/weak cell adhesion [9–12]. The present study suggests that the native hornet silk, which is a mixture of Vssilk1–4, has cytocompatibility similar to B. mori silk, with malleable mechanical properties resulting from its coiled-coil molecular structure [23]. In addition, the native hornet silk film has flexibility and mechanical properties suitable for surgical handling in wet conditions [22], where biomaterials must show their performance. Individually, the component proteins generate unique materials with properties distinct from the native composite material. Of the films generated from the recombinant Vssilk proteins, those generated from Vssilk1 or 2 are excellent candidates for silk-derived scaffold materials, with high-yield productivity, structural and mechanical properties similar to the native hornet silk, but with improved cell adhesion activity comparable with TCPS. Moreover, there is a possibility that the cytocompatibility of Vssilk scaffolds is adjustable in different combinations of the four Vssilk proteins. The present authors are currently investigating characteristic changes, depending on the combination of Vssilk proteins and developing methods to fabricate these proteins into various threedimensional forms suitable for cell scaffolds.
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Full-length versions of the four V. simillima hornet silk proteins, Vssilk1–4, were recombinantly produced in E. coli at high levels.
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Films generated from solutions of each of the Vssilk proteins mimic the protein secondary structure and mechanical property of native hornet silk films. In contrast to silk films generated from reconstituted native silkworm or hornet silk proteins or those generated from recombinant Vssilk3 and 4, films generated from Vssilk1 or 2 are hydrophobic and have high pI. The high pI of these proteins results in a net positive charge on the surface of films in physiological conditions and results in high fibroblast adhesion to these materials. The recombinant yield, mechanical properties and cell adhesion properties suggest that several of the recombinant hornet silk proteins may be valuable materials from which to generate protein scaffolds for tissue culture applications.
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Disclosures
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The authors confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.
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Acknowledgements
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The authors would like to thank Alagacone Sriskantha from Ecosystem Sciences, CSIRO, for helping with the production and purification of recombinant silk proteins; and Hiroko Saito from Silk Materials Research Unit, NIAS for helping with the film surface analysis. This work was financially supported in part by JSPS Grant-in-Aid for Scientific Research (C) 24580086 and JSPS Grant-in-Aid for JSPS Fellows 25-10369.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.05. 013.
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Recombinant production and film properties of full-length hornet silk proteins
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Yusuke Kambe a, Tara D. Sutherland b, Tsunenori Kameda a,⇑ a b
Silk Materials Research Unit, National Institute of Agrobiological Sciences (NIAS), 1–2 Owashi, Tsukuba, Ibaraki 305-8634, Japan Ecosystem Sciences, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Clunies Ross St., Acton, ACT 2601, Australia
a r t i c l e
i n f o
Article history: Received 20 December 2013 Received in revised form 1 May 2014 Accepted 15 May 2014 Available online xxxx Keywords: Biomimetic material Fourier transform infrared spectroscopy Mechanical test Surface analysis Isoelectric point
a b s t r a c t Full-length versions of the four main components of silk cocoons of Vespa simillima hornets, Vssilk1–4, were produced as recombinant proteins in Escherichia coli. In shake flasks, the recombinant Vssilk proteins yielded 160–330 mg recombinant protein l 1. Films generated from solutions of single Vssilk proteins had a secondary structure similar to that of films generated from native hornet silk. The films made from individual recombinant hornet silk proteins had similar or enhanced mechanical performance compared with films generated from native hornet silk, possibly reflecting the homogeneity of the recombinant proteins. The pH-dependent changes in zeta (f) potential of each Vssilk film were measured, and isoelectric points (pI) of Vssilk1–4 were determined as 8.9, 9.1, 5.0 and 4.2, respectively. The pI of native hornet silk, a combination of the four Vssilk proteins, was 4.7, a value similar to that of Bombyx mori silkworm silk. Films generated from Vssilk1 and 2 had net positive charge under physiological conditions and showed significantly higher cell adhesion activity. It is proposed that recombinant hornet silk is a valuable new material with potential for cell culture applications. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
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1. Introduction
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Silks are fibrous protein materials produced by a large number of arthropods [1]. The silk of the domesticated silkworm Bombyx mori has been used in medicine for centuries as surgical suture and, more recently, degummed silk has been FDA-approved for clinical use [2]. The native silk proteins have been reconstituted from natural silk and processed into films, meshes, gels and sponges for applications such as scaffolds for tissue engineering [3–5]. Besides their mechanical properties, these materials possess other characteristics essential for scaffolds: they are biodegradable and non-cytotoxic, and they generate low-immunity and low-inflammatory reactivities [3,6–8]. However, silk scaffolds reportedly show low/weak cell adhesion [9–12], because most of the silk proteins have no cell adhesive amino acid sequences. Additionally, transgenic technologies, aimed at producing a homogeneous supply of the silk proteins and enabling the modification of the proteins towards specific biocompatibility and functional requirements, have been unsuccessful in generating commercial scale processes. The expression efficiency of proteins in recombinant B. mori silkworms remains low (at most 15%) [13], and the large (>10 kbp) and repetitive silk gene sequences
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⇑ Corresponding author. Tel./fax: +81 29 838 6213. E-mail address: [email protected] (T. Kameda).
have limited other recombinant systems to the expression of fragments of the silk proteins [14,15]. In addition to the well-known cocoons produced by silkworms and webs produced by spiders, social hymenopterans, including honeybees, ants and hornets, also produce ample amounts of silk. The principal molecular structure of the social hymenopteran silk is a-helices, frequently in a coiled-coil conformation [16], a molecular structure distinct from the b-crystalline structures that dominate the silks of silkworms and spiders. Full-length silk genes from a number of social hymenopteran species have been identified and cloned [15,17,18]. The small size (1 kbp) and comparatively low levels of repetition in these genes have allowed recombinant production of full-length honeybee silk proteins, AmelF1–4, in E. coli at high yield and potentially on a large scale [19]. Silk cocoons produced by larvae of yellow hornets, Vespa simillima (Fig. 1), consist of four major proteins: Vssilk1–4 [18,20]. Vssilk1–4 are homologous to AmelF1–4 and are also small (30–70 kDa) and non-repetitive, in contrast to silk proteins of silkworms and spiders. Unlike ant and honeybee silks, V. simillima silk (hornet silk) can be dissolved in concentrated salt solutions (e.g. 9 M LiBr) [20,21], probably owing to the lower presence of e-(cglutamyl)-lysine crosslinks [21]. The solubility of the hornet silk in salt solutions would lead its reconstitution into highly pure products, because the salt can be removed by simple methods such as dialysis. Reconstituted hornet silk can be readily processed into
http://dx.doi.org/10.1016/j.actbio.2014.05.013 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Please cite this article in press as: Kambe Y et al. Recombinant production and film properties of full-length hornet silk proteins. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.05.013
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Fig. 1. Photograph of silk cocoons of yellow hornets (Vespa simillima).
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transparent films that are flexible and with mechanical properties suitable for surgical handling [22]. The mechanical properties of these hornet silk films include a breaking tensile strain of 100%, a maximum tensile strength of 170 MPa and tensile modulus of 5.5 GPa [23], values superior to those of films fabricated from reconstituted B. mori silk proteins [24,25]. As found in other silks, hornet silk is dominated by the small amino acids Ala and Ser (35% and 23%, respectively). However, the four hornet silk proteins also contain significant levels (16%) of charged amino acids (Arg, Asp, Glu and Lys). The charged amino acid composition of each of the proteins is different, resulting in unique predictions for the isoelectric point (pI) for each protein (ranging from 4.4 to 10.3 [18]). In contrast, the predicted pI of the four honeybee silk proteins, AmelF1–4, are similar (from 5.1 to 5.8 [18]). B. mori silk fibroin, which is composed of a heavy chain (H-chain) and a light chain (L-chain), has a pI of around five [8]. Hence, the hornet silk is composed of a complex assembly of multi-silk proteins, each with different physicochemical properties. These findings suggest that the hornet silk can be expected to be fabricated into highly pure scaffolds that provide specific physical and chemical environments for various types of cells, depending on varied combinations of the four Vssilk proteins. However, the homologous preparation of each of the four silk proteins has not been established, and their characteristics remain unclear. The goal in the present study was to produce full length versions of the four Vssilk proteins in E. coli in order to investigate the structure, mechanical performance and physicochemical surface properties of materials generated from the individual proteins. Cast films from each Vssilk protein were generated and compared with films generated from reconstituted native hornet silk and reconstituted B. mori silk. The results indicated that films generated from recombinant Vssilk proteins have specific surface wettability and charge properties conducive to cell adhesion to materials.
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2. Materials and methods
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2.1. Recombinant production of four hornet silk proteins
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To prepare vectors for the expression of Vssilk1–4, the four hornet silk gene sequences (accession no. AB537885, AB537886, AB537887 and AB537888) without signal peptides were amplified by polymerase chain reaction (PCR) from the previously reported cDNA clones [18]. The four silk proteins have similar tri-block architecture, and the full amino acid sequence of the expressed proteins is shown in Supplementary Data Fig. 1. The codon for the first amino acid (Ser) of the native Vssilk2 protein was replaced by a codon for Gly to enable cloning into the vector NcoI site. The
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oligonucleotide primers used to amplify cDNA prior to cloning into expression vectors were as follows, with NcoI, NdeI and BamHI sites underlined and sequences that match the cDNA italicized: Vssilk1: GGATTCCC ATG GGG CCA TCA AGG TTG TCT GAA ACC and CGGCCATATG TTA TTA GGC GCT GCT ACT ACT CGA AGA GGC; Vssilk2: GGATTCCC ATG GCG GCA GCG ATC GCA TCG GCT CGA and CGGCGGATCC TTA TTA GTT TCC GGA TGC GCT GCG AGA ACT; Vssilk3: GGATTCCC ATG GCA GAA AGC TCA AGC TCA AGC TCC and CGGCGGATCC TTA TTA CCA ATC CTG GGA CCA GAT ATT CTG; Vssilk4: GGATTCCC ATG GAC AGA TCG TGG GCA GCA TCG GAT and CGGCGGATCC TTA TTA GTT TAA GAT CAG CGA GCT AGT TCT. The PCR products were digested with appropriate restriction enzymes (Vssilk1: NcoI and NdeI; and Vsilk2–4: NcoI and BamHI; New England Biolabs) and cloned into the pET14b expression vector (Novagen) using T4 DNA ligase (New England Biolabs). The constructs were verified by restriction digest and DNA sequencing before expression. E. coli strain Rosetta 2 (DE3) competent cells (Novagen), transformed with the vector DNA, were plated onto LB agar containing 150 lg ml 1 ampicillin and 50 lg ml 1 chloramphenicol. After overnight incubation at 37 °C, recombinant colonies were transferred to Overnight Express Instant TB medium (Novagen) with 10% (v/v) glycerol in shake flasks and then incubated for 48 h at 25 °C or 24 h at 28 or 31 °C. Cultures were pelleted by centrifugation at 10,000g for 10 min at 4 °C and stored at 20 °C until use.
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2.2. Analysis of expression levels of recombinant hornet silk proteins
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The soluble fraction and inclusion bodies from recombinant E. coli were separated using BugBuster Master Mix (Novagen) according to the manufacturer’s protocol. Inclusion bodies were further purified after the cells were resuspended in five times their volume of the BugBuster reagent and incubated for 30 min at room temperature (RT). After centrifugation at 10,000g for 10 min at 4 °C, the pellet was washed with five times its volume of the BugBuster reagent, followed by washing in five times its volume of the BugBuster reagent diluted tenfold with phosphate-buffered saline (PBS). The purified inclusion bodies were resuspended in 9 M LiBr aqueous solution, incubated at 70 °C for 30 min and centrifuged at 10,000g for 10 min at 4 °C to remove bacterial proteins. The solutions of essentially pure Vssilk1, Vssilk2, Vssilk3 or Vssilk4 in 9 M LiBr were filtered through a 0.22-lm membrane (Merck) to remove insoluble matter. The solutions were then diluted with four times volume of reverse osmosis (RO) water, enough not to affect the following concentration analysis and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [20]. BCA protein assay kits (Pierce) were used to determine the concentration of the protein and to calculate expression levels. The protein composition of the Vssilk1–4/LiBr solutions was analyzed by SDS-PAGE on Mini-PROTEAN TGX gels (Any kD; Bio-Rad) under a reducing condition with the intensity of protein bands measured by ImageJ (NIH).
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2.3. Films of native hornet silk, recombinant Vssilk 1–4 and silk fibroin
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Cocoons of V. simillima were collected from their nest and dissolved in 9 M LiBr solution by stirring for 1 h at 37 °C, followed by filtering through a 0.22-lm membrane to remove the nest papers adhering to the cocoons. B. mori silk/LiBr solution was prepared by dissolving degummed fibers of cocoons in 9 M LiBr solution at RT overnight. The recombinant Vssilk1–4, native hornet silk and B. mori silk / 9 M LiBr solutions were dialyzed against RO water, using cellulose dialysis membranes (MWCO, 6000–8000; Spectrum Laboratories) for 3 days at 4 °C, changing the water every 10–14 h, resulting in
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the almost complete removal of Li+ and Br ions [26]. During the dialysis, the solutions, except the silk fibroin solution, tuned into a turbid gel or resulted in a precipitate. The silk gels, precipitates or aqueous solutions were freeze-dried. The resultant dried silk proteins were immediately weighed and dissolved in hexafluoroisopropyl alcohol (HFIP) by stirring at RT overnight to prepare 1% (w/v) silk protein/HFIP solutions. Films of recombinant Vssilk1, Vssilk2, Vssilk3 and Vssilk4, native hornet silk and silkworm silk fibroin were prepared by spreading the protein/HFIP solution over the following plates and dishes at 100 ll cm 2: borosilicate glass plates (24 60 mm; Matsunami Glass) for structural and surface analyses; 50-mm polystyrene dishes (Eiken Chemical) for tensile testing; and 24well polystyrene plates (IWAKI) for cell culture. After spreading the solution, the plates and dishes were air dried at RT for 24 h. Films on the plates and dishes were then immersed in 80% (v/v) methanol at RT for 1 h, to make them water insensitive, and air dried at RT for 24 h. Following this, the films were sterilized by submerging in 70% (w/w) ethanol for 20 min, rinsed twice with autoclaved ultrapure water, and air dried at RT for 24 h. The resultant silk films were photographed under RT air conditions using a digital camera (EOS-10D; Canon).
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2.4. Fourier transform infrared spectroscopy (FTIR)
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FTIR analysis of the silk films (as-cast, after treatment with 80% (v/v) methanol, and after sterilization with 70% (w/w) ethanol) was performed using a spectrometer (FT/IR-620 and IRT-30; Jasco), equipped with a single-reflection Ge ATR attachment (ATR-30-G45; Jasco). The instrument was continuously purged with nitrogen gas to remove atmospheric water vapor. The spectra were recorded with an accumulation of 128 scans at a resolution of 4 cm 1.
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2.5. Mechanical testing of silk films
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For tensile testing of the silks, the films were gently peeled from the polystyrene dishes and cut into strips 2.0 mm wide and 30.0 mm long. The thickness of the films was estimated to be 4.9 ± 1.0 lm by electron microscopy (TM-1000; Hitachi). The films were stored in a desiccators until use. The tensile properties of the dry specimens were measured using a tensile tester (EZ test; Shimadzu), equipped with a 5 N load cell, at a rate of 5 mm min 1. The gauge length was set at 20 mm. Tests were conducted in air at 24–26 °C and 41–48% relative humidity. From the stress/strain curves, maximum stress, breaking stress, breaking strain and toughness were determined. The tensile moduli were determined from the initial slope of the stress/strain curves at a strain of 1– 1.2%. Nine different samples were tested (n = 9).
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2.6. Measurement of surface properties of silk films
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Static water contact angles on the films were determined by the sessile drop method, using a contact angle analyzer (FTA188; First Ten Angstroms) at RT. A droplet (3.5 ll) of ultrapure water was placed on the film surface, and the time-dependent changes of the contact angle were recorded for 90 s. The water contact angle was determined as the intercept contact angle (at 0 s) of a line fitted between 30 and 60 s. Eight different areas were analyzed (n = 8). The zeta (f) potential of each film was measured using a fpotential analyzer (ELS-8000; Otsuka Electronics), equipped with the cell unit for flat plate samples, in 5 mM sodium phosphate buffer and 5 mM NaCl at 25 °C. The pH of the buffer was adjusted to 3.00, 4.00, 5.00, 6.00, 7.00, 8.00 and 9.00 with HCl and NaOH aqueous solutions. A sample was used to measure f potential at the seven pH by changing the streaming solution. Three different
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samples were used (n = 3). Curve fitting was done using two- or three-degree polynomial regression for each sample, and the f potential at pH 7.4 and pI of the sample were calculated.
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2.7. Cell culture on silk films
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The ability of the silken films to promote cell attachment was measured after NIH3T3 murine fibroblasts in Eagle’s minimum essential medium (Nissui Parmaceutical) supplemented with 10% (v/v) fetal bovine serum (Gibco), 10 ng ml 1 kanamycin (Gibco), 0.2% sodium bicarbonate (Gibco) and 2 mM l-Glu (Gibco) were seeded onto the films prepared in 24-well cell culture plates at 5.0 103 cells cm 2. The films and cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2 for up to 24 h, and cell morphology and distribution on the films were observed using a phase-contrast inverted microscope (IX-70, Olympus), equipped with a cooled CCD camera (TCH5.0ICE; Xintu Photonics). The number of cells that had adhered to the silk films was determined using the previously described lactate dehydrogenase (LDH) assay [27]. Briefly, after incubation, the cells were washed twice with PBS. Washed cells were then dissolved by overnight incubation in 0.5% (w/v) Triton X-100 (Research Organics) in PBS at 4 °C. The LDH activity of the dissolved cells was measured from changes in the absorbance at 340 nm that are attributed to the kinetics of nicotinamide adenine dinucleotide-consuming reactions. The adherence of fibroblasts cultured on tissue culture polystyrene (TCPS; IWAKI) were measured as a positive control. Four different films were used for each sample point (n = 4).
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2.8. Statistical analysis
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The tensile and surface properties (water contact angle, f potential at pH 7.4, and pI) of the various films were compared using one-way ANOVA, followed by Dunnett post hoc analysis, using the measurements from the native hornet silk films as the control. The effects of the type of film and culture time on cell number were analyzed using two-way ANOVA, followed by Dunnett post hoc comparisons with the native hornet silk film as the control. A value of P < 0.05 was considered significant. In all figures, asterisks indicate statistical differences from the native hornet silk film.
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3. Results
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3.1. Production of recombinant hornet silk proteins and analysis of film impurities
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The four full-length hornet silk proteins (Vssilk1–4) were successfully synthesized after expression from transgenic E. coli in shake flasks. At all culture temperatures investigated in this study (25, 28, and 31 °C), SDS-PAGE (data not shown) indicated that the silk proteins were more highly expressed in inclusion bodies than in the soluble form. Highest levels of expression were achieved after 48 h expression at 25 °C (Vssilk1, 3 and 4), and after 24 h expression at 31 °C for Vssilk2. Proteins appeared not to be solubilized from the inclusion bodies with SDS, a detergent commonly used in molecular biology to analyze proteins, and therefore only slight amount of the proteins were observed on SDS-PAGE gels without further treatment. The inclusion bodies could be solubilized in 9 M LiBr, an ionic solution used to reconstitute native hornet and silkworm silk. Under these conditions, 330, 170, 160 and 330 mg culture medium of Vssilk1, 2, 3, and 4 proteins, respectively, were achieved at a high purity (>90%; Fig. 2). After removal of LiBr and processing into films, as described in Section 2, solid-state 13C CP/MAS nuclear magnetic resonance (NMR) analysis of recombinant hornet silk films generated from these proteins (Supplementary Data Fig. 2) showed that
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Fig. 2. SDS-PAGE of recombinant hornet silk proteins Vssilk1–4. Lane numbers correspond to recombinant V. simillima hornet silk proteins Vssilk1–4. N indicates the lane for native hornet silk proteins. NovexÒ Sharp Protein Standard (Invitrogen) was used as molecular weight markers (M).
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no BugBuster detergent, LiBr or HFIP derived impurities were contained in the final recombinant films. As shown in Fig. 3, the silk films were generally smooth and transparent, with the Vssilk1 film slightly opaque.
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3.2. Molecular structure of proteins in silk film
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The secondary structures of the proteins in the various recombinant silk films were compared with those in films generated from
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native hornet and silkworm silk using FTIR (Fig. 4). In FTIR spectra, the signal at 1650 and 1624 cm 1 can be attributed to a-helix and b-sheet conformations, respectively. Initially, all the silk films contained a predominantly a-helix structure (Fig. 4a). Submersion in an 80% (v/v) methanol and drying (Fig. 4b) induced a conformational transition from the a helix to the b sheet, particularly in the B. mori silk film. In contrast, the native hornet silk and recombinant Vssilk films consisted of coexisting a-helix and b-sheet conformations, with the Vssilk4 film being comparatively rich in b-sheets in comparison with the other hornet silk films. Immersion in 70% (w/w) ethanol and drying (Fig. 4c), which served to sterilize the films, promoted further transition to b-sheet structure in all films. However, all hornet silk films maintained both a-helix and b-sheet structures. In comparison with the native hornet silk film, the recombinant Vssilk1, 2 and 3 films were rich in a-helices, whereas the Vssilk4 film was rich in b-sheet structure.
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3.3. Tensile properties of silk films
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The mechanical properties of the sterilized silk films in dry conditions are shown in Table 1. All the recombinant Vssilk films showed mechanical properties comparable with or superior to the native hornet silk film, with the exception of the breaking strain of the Vssilk2 film. In particular, the films of Vssilk3 or 4 proteins were more ductile than the native hornet silk film, without a decrease in tensile modulus. The B. mori silk film was harder and brittler than the hornet silk films, showing the lowest breaking strain and toughness, but the highest tensile modulus among the films tested in this study.
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3.4. Physicochemical properties of silk films
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The water contact angles on the various silk films are shown in Table 2. The contact angle measurements for the Vssilk1 and 2 films are significantly higher than those for the native hornet silk film, indicating that the surface of the Vssilk1 or 2 films are more hydrophobic. There are no significant differences between either the Vssilk3 or Vssilk4 and native hornet silk films. The B. mori silk film had a more hydrophilic surface than any of the hornet silk films. Fig. 5 shows the f potential of the silk films in different pH environments. The profile of f potential across different pH
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Fig. 3. Photographs of HFIP cast silk films after treatments with 80% (v/v) methanol and 70% (w/w) ethanol followed by washing with ultrapure water. Scale bar = 2 cm.
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Fig. 4. FTIR spectra of the amide I region of films of recombinant hornet silk and reconstituted native hornet and silkworm silks. The signal at 1650 and 1624 cm 1 are attributed to the a-helix and b-sheet conformations, respectively. In all figures, dotted lines indicate the spectrum for native hornet silk films. (a) Cast film, (b) after treatment with 80% (v/v) methanol, and (c) after sterilization with 70% (w/w) ethanol followed by washing with ultrapure water.
Table 1 Tensile properties of silk films. Film Hornet silk
Native Vssilk1 Vssilk2 Vssilk3 Vssilk4
B. mori silk
Maximum stress (MPa)
Breaking stress (MPa)
Breaking strain (%)
Toughness (mJ)
Tensile modulus (GPa)
54 ± 6 62 ± 8 56 ± 17 62 ± 5 74 ± 17⁄⁄
45 ± 12 56 ± 9 55 ± 17 56 ± 4 73 ± 17⁄⁄⁄
3.8 ± 0.6 4.4 ± 0.5 3.1 ± 0.6⁄ 4.8 ± 0.5⁄⁄ 4.6 ± 0.7⁄
0.25 ± 0.04 0.32 ± 0.07 0.21 ± 0.12 0.38 ± 0.08⁄⁄ 0.38 ± 0.07⁄⁄
2.2 ± 0.4 2.1 ± 0.6 2.5 ± 0.7 2.3 ± 0.4 2.4 ± 0.4
57 ± 19
57 ± 19
2.3 ± 0.7⁄⁄⁄
0.13 ± 0.07⁄⁄
2.9 ± 0.6⁄
Data shown in the form: mean ± SD (n = 9). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄P < 0.05, ⁄⁄P < 0.01, ⁄⁄⁄P < 0.001).
Table 2 Biophysical properties of silk films. Film Hornet silk
B. mori silk
Water contact angle (deg) Native Vssilk1 Vssilk2 Vssilk3 Vssilk4
73.1 ± 4.0 84.6 ± 5.3⁄⁄⁄ 90.7 ± 5.3⁄⁄⁄ 74.3 ± 9.2 70.0 ± 5.2 63.1 ± 1.6⁄⁄
f potential at pH 7.4 (mV) 19.5 ± 6.7 +6.8 ± 1.8⁄⁄⁄ +4.5 ± 3.8⁄⁄⁄ 14.1 ± 5.1 29.9 ± 8.4 25.1 ± 1.9
Empirical pI
Calculated pI
4.7 ± 0.1 8.9 ± 0.5⁄⁄⁄ 9.1 ± 1.9⁄⁄⁄ 5.0 ± 0.1 4.2 ± 0.0 3.5 ± 0.4
ND 10.29 [14] 10.18 [14] 5.76 [14] 4.38 [14] 4.39 (H-chain) [8] 5.06 (L-chain) [8]
Data shown in the form: mean ± SD (n = 8 for water contact angle; n = 3 for f potential and pI). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄⁄P < 0.01, ⁄⁄⁄P < 0.001). ND: not determined.
Fig. 5. f potential in different pH environments of (A) native or recombinant hornet silk films and (B) B. mori silk film. Curve fitting was done using two- or three-degree polynomial regression. Data are shown in the form: mean ± SD (n = 3).
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Fig. 6. (A) Photographs and (B) quantitative data of fibroblast attachment to recombinant hornet silk protein, native hornet silk and native B. mori silk films after 12 and 24 h. Scale bar = 100 lm in Fig. 6A. For Fig. 6B, the vertical axis indicates the proportion of cells attached relative to the number of cells attached to TCPS dishes. Data are shown in the form: mean ± SD (n = 4). Asterisks indicate significant differences analyzed with Dunnett’s test with the native hornet silk group as the control, following one-way ANOVA (⁄P < 0.05, ⁄⁄P < 0.01).
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environments of the Vssilk1 and 2 films were significantly different from those of the native hornet silk film and Vssilk3 and 4 films; the Vssilk1 and 2 films tended to show positive charges throughout the pH 3–9 range, whereas the others had pI (pH at f potential = 0) between pH 4 and 5 (Fig. 5A). Hence, the surface of Vssilk1 and 2 films had a slightly positive charge under physiological conditions (pH 7.4), whereas the native hornet silk films and Vssilk3 and 4 films were strongly negatively charged (Table 2). The B. mori silk film had pI between the pH 3 and 4 (Fig. 5B) and was also strongly negatively charged at pH 7.4 (Table 2). The empirical pI determined from regression curves corresponded approximately to pI calculated using the ProtParam tool (http://web.expasy.org/protparam/) (Table 2).
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3.5. Cell adhesion to silk films
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Cell morphology and distributions of NIH3T3 fibroblasts on the various silk films after 12 and 24 h post-seeding are shown in Fig. 6A. The surface of the silk films was non-uniform; particles (native hornet silk and Vssilk2), cracks (native hornet silk and Vssilk3) and rough surface (Vssilk1 and B. mori silk) appeared. Although all the silk films had a wavy surface, this pattern was also seen on the surface of the TCPS (data not shown). However, no tendencies of cell adhesion, such as distribution and orientation, were observed depending on these surface structures of the silk films. In contrast, cell morphologies varied, depending on the type of silk proteins. Fibroblasts tended to gather on the native hornet silk, Vssilk3 and B. mori silk films, whereas cells spread out on the films generated from Vssilk1, 2 and 4 proteins. A comparison of the number of the cells attached to the various silk films is shown in Fig. 6B. The cell number is displayed as the ratio of attached cells in comparison with the number observed attached to TCPS after the equivalent time period. The number of
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cells on the Vssilk1 or 2 films was comparable to that on TCPS and significantly larger than that on either native hornet silk films or B. mori silk films after both 12 and 24 h in culture. Cell attachment to the Vssilk3 and 4 films was not significantly different from that observed on native hornet silk films or B. mori silk films after 24 h in culture.
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4. Discussion
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In this work, recombinant full-length hornet silk proteins, Vssilk1–4, were produced in shake flasks from recombinant E. coli at yields of 160–330 mg from a liter of culture medium. Given that yield gains can be expected if expression is optimized in fermentation facilities rather than shake flasks, the yields of recombinant Vssilk rival those seen in other systems, including honeybee silk proteins at 0.2–2.5 g l 1 [19], native-sized spider silk protein at 0.5–2.7 g l 1 [28], a partial-length spider silk at 0.36 g l 1 [29] and full-length mantis fibroins at 40–400 mg l 1 [30]. The high yields of recombinant hornet silk production probably stem from the smaller size and low repetition in the sequences of silk genes. Standard analysis of proteins involves SDS-PAGE to determine protein size and purity. Previously, the honeybee silks were purified from inclusion bodies using SDS [19], the detergent promoting the folding of the silk proteins into a-helices [31]. However, it was difficult to dissolve the inclusion bodies in the present study in SDS. Saturated LiBr, a salt commonly used to reconstitute native silkworm and hornet silks, did solubilize the inclusion bodies, and the solution contained highly pure Vssilk proteins (Fig. 2). Concentrated LiBr solution completely solubilizes hornet silk [20,21], allowing insoluble impurities to be removed by centrifugation and filtration. Following removal of impurities, the silk proteins could be recovered from solution after inducing their precipitation
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by the removal of Li+ and Br ions after dialysis against RO water [23]. The silk proteins could then be recovered from the solution, freeze dried and processed into materials that did not have residual detergent or solvent present (Supplementary Data Fig. 2). It is generally known that silkworm silk proteins dissolved in HFIP solution tend to form helix conformations, and this conformation is maintained in films fabricated from these solutions [32]. The helix conformation in these silk products is transformed into b-sheet by treatment in alcohol [33–35]. The dominant molecular structure in native silkworm silk is b-sheet. Similarly, the same molecular changes in silkworm silk film were found in this study. The molecular structure of proteins in native hornet silk is different from that found in silkworm silk. The molecular architecture of the Vssilk1–4 proteins involves a central Ala-rich region that adopts an a-helical conformation, and Ser-rich regions at both ends that adopt the b-sheet conformation [18,23,36]. Bioinformatics predicts that a significant proportion of the central a-helices in the Ala-rich region forms coiled-coil structures [18,23], similar to the coiled-coil structures observed in honeybee silk. As with B. mori silk films, all the films cast from hornet proteins in HFIP solutions were rich in a-helices (Fig. 4a), and methanol and ethanol treatments promoted the formation of b-sheets (Fig. 4b and c). However, in contrast to the dramatic change in b-sheets observed in B. mori silk films, alcohol treatments induced a smaller change, and the final films maintained the coexistence of a-helices and b-sheets similar to that observed in native hornet silk films. Within the recombinant hornet silk films, the proportion of a-helix to b-sheet varied; a-helices dominated films of Vssilk1–3, whereas b-sheets dominated the Vssilk4 films (Fig. 4c). Previously, the fraction of coiled-coil structures in Vssilk1–3 was determined as 60%, but only 50% in the Vssilk4 protein [37]. The agreement between these results suggests that a-helices observed in the recombinant Vssilk films are predominantly in coiled-coil structures that are preserved after the immersion in aqueous alcohol. It is likely that the coiled-coil structure prevents transition of a-helices to b-sheets. Overall, the structural analysis indicates that the molecular structure in films generated from single recombinant Vssilk proteins mimics that in native hornet silk film with coexisting coiled-coil/a-helices and b-sheets. The recombinant Vssilk films had mechanical properties either equaling or surpassing those of the native hornet silk and B. mori silk films (Table 1). The retention of mechanical performance in films generated from a single Vssilk protein agrees with previous work by Sutherland et al. [38], which found that fibers generated from a single recombinant honeybee silk protein, AmelF3, have mechanical properties similar to fibers generated from all four of the proteins found in native honeybee silk. The finding that a single Vssilk protein can mimic the structural and mechanical properties of native hornet silk supports the conclusion that the four paralogous silk genes have been conserved for 150 million years in each social hymenopteran lineage, as a result of their protein products playing a critical role in the process of natural silk fabrication rather than in conferring properties to the end product [38]. Physicochemical surface properties of the Vssilk films varied: films from Vssilk3 and 4 possessed surface properties similar to the native hornet silk and B. mori silk fibroin, whereas the films generated from Vssilk1 and 2 were more hydrophobic and with positive surface charges (Fig. 5 and Table 2). These characteristics, particularly f potential on the silk film surface, seemed to be influenced primarily by the amino acid composition of the silk proteins, as the predicted pI of the silk proteins corresponded well to the experimental results. It is widely accepted that cell adhesion to materials is strongly influenced by the materials’ surface properties, including roughness, wettability and charge [39–48]. Since the primary structure of the Vssilk1–4 contains no specific integrin-binding motifs such as RGD, PHSRN and YIGSR (Supplemen-
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tary Data Fig. 1), the physicochemical surface properties of the silk films play an important role in promoting cell attachment (Fig. 6). Of the properties, the surface topology of the films was unlikely to heavily affect cell adhesion, because no strong tendencies were observed, depending on the topology (Fig. 6A). As for cell numbers in this study (Fig. 6B), the best cell adhesion was observed on the hydrophobic, positively charged films generated from Vssilk1 and 2 proteins. Generally, these properties have conflicting effects on cell adhesion, with other studies reporting inhibition of cell attachment to surfaces with water contact angle of >90° [41,47] and enhancement of cell adhesion to materials with positive charges [45,46]. In silken materials, silks with low pI values, i.e. negatively charged under physiological conditions, generally show weaker cell adhesion than silks with high pI values [8]. The present authors propose that the specific positive surface charges on the surface of films generated from Vssilk1 or 2 enhance cell adhesion as a result of cell–substrate interactions medicated by adsorbed negatively charged extracellular matrix proteins such as type I collagen (pI 5.46) and fibronectin (pI 5.60). Interestingly, among the recombinant hornet silk films, only fibroblasts on Vssilk3 film showed morphology similar to the cells on the native hornet silk and B. mori silk films (Fig. 6A). Although the four silk proteins in social hymenopteran silks are considered to be expressed and produced at approximately equivalent levels [49,50] to assemble into a hetero-tetrameric coiled-coil [16,36], it is inferred that Vssilk3 proteins were selectively exposed to the surface of the native hornet silk film, which resulted in similar wettability, charge and cytocompatibility of both film surfaces (Table 2 and Fig. 6). Polymer-based scaffolds can be classified into synthetic (e.g. poly(glycolic acid) and poly(lactic acid)) and natural polymers (e.g. collagen and fibrin) [51]. The synthetic polymers provide suitable bulk, flexibility and mechanical properties, but they generally lack chemical cues to stimulate cell adhesion and proliferation [51,52] and require harsh chemicals for polymerization and modification [51]. In contrast, natural polymers, particularly collagen, show biomimetic structure and cytocompatibility [51,53], but they need chemical treatments to generate suitable mechanical properties [52], and there remains a risk of immunity/infection hazards from residual antigens [53]. B. mori silk minimizes these disadvantages, but it shows only low/weak cell adhesion [9–12]. The present study suggests that the native hornet silk, which is a mixture of Vssilk1–4, has cytocompatibility similar to B. mori silk, with malleable mechanical properties resulting from its coiled-coil molecular structure [23]. In addition, the native hornet silk film has flexibility and mechanical properties suitable for surgical handling in wet conditions [22], where biomaterials must show their performance. Individually, the component proteins generate unique materials with properties distinct from the native composite material. Of the films generated from the recombinant Vssilk proteins, those generated from Vssilk1 or 2 are excellent candidates for silk-derived scaffold materials, with high-yield productivity, structural and mechanical properties similar to the native hornet silk, but with improved cell adhesion activity comparable with TCPS. Moreover, there is a possibility that the cytocompatibility of Vssilk scaffolds is adjustable in different combinations of the four Vssilk proteins. The present authors are currently investigating characteristic changes, depending on the combination of Vssilk proteins and developing methods to fabricate these proteins into various threedimensional forms suitable for cell scaffolds.
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5. Conclusions
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Full-length versions of the four V. simillima hornet silk proteins, Vssilk1–4, were recombinantly produced in E. coli at high levels.
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Films generated from solutions of each of the Vssilk proteins mimic the protein secondary structure and mechanical property of native hornet silk films. In contrast to silk films generated from reconstituted native silkworm or hornet silk proteins or those generated from recombinant Vssilk3 and 4, films generated from Vssilk1 or 2 are hydrophobic and have high pI. The high pI of these proteins results in a net positive charge on the surface of films in physiological conditions and results in high fibroblast adhesion to these materials. The recombinant yield, mechanical properties and cell adhesion properties suggest that several of the recombinant hornet silk proteins may be valuable materials from which to generate protein scaffolds for tissue culture applications.
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Disclosures
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The authors confirm that there are no known conflicts of interest associated with this publication, and there has been no significant financial support for this work that could have influenced its outcome.
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Acknowledgements
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The authors would like to thank Alagacone Sriskantha from Ecosystem Sciences, CSIRO, for helping with the production and purification of recombinant silk proteins; and Hiroko Saito from Silk Materials Research Unit, NIAS for helping with the film surface analysis. This work was financially supported in part by JSPS Grant-in-Aid for Scientific Research (C) 24580086 and JSPS Grant-in-Aid for JSPS Fellows 25-10369.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.05. 013.
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References
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