Sterilized Recombinant Spider Silk Fibers of Low Pyrogenicity

Biomacromolecules 2010, 11, 953–959

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Sterilized Recombinant Spider Silk Fibers of Low Pyrogenicity My Hedhammar,*,† Hanna Bramfeldt,† Teodora Baris,‡ Mona Widhe,† Glareh Askarieh,§ Kerstin Nordling,† Sonja von Aulock,‡ and Jan Johansson† Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Centre, SE-751 23 Uppsala, Sweden, Biochemical Pharmacology, University of Konstanz, Universitaetsstr. 10, 78464 Konstanz, Germany, and Department of Chemistry, Oslo University, 1033 Blindern, 0315 Oslo, Norway Received December 9, 2009; Revised Manuscript Received February 15, 2010

We have recently shown that it is possible to recombinantly produce a miniature spider silk protein, 4RepCT, that spontaneously self-assembles into mechanically stable macroscopic fibers (Stark, M.; Grip, S.; Rising, A.; Hedhammar, M.; Engstrom, W.; Hjalm, G.; Johansson, J. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 2007, 8 (5), 1695-1701). When produced as a soluble fusion protein (with thioredoxin) in Escherichia coli, the spider silk protein can be subjected to several purification steps without aggregating. Here, combined purification and endotoxin removal is achieved using a simple cell wash procedure, protein affinity purification, and LPS depletion. No toxic chemicals were included in the process and the protein retained its ability to self-assemble into fibers. With this method, fibers with pyrogenicity corresponding to less than 1 EU/mg could be recovered. Moreover, the fibers could be sterilized through autoclaving with retained morphology, structure, and mechanical properties. This implies that this recombinant silk is suitable for usage as biomaterial, which is further supported by data showing that the fibers allow growth of human primary fibroblasts.

Introduction Spider silk appears to exhibit all desired properties for usage as a biomaterial: strength and elasticity combined with biocompatibility.2,3 The proteins that build up spider silk have a tripartite composition: an N-terminal nonrepetitive domain, a highly repetitive central part composed of poly-Ala/Gly-rich cosegments, and a C-terminal nonrepetitive domain.4 Recently we showed that a miniature spider silk protein composed of four poly-Ala/Gly-rich cosegments and the C-terminal domain, 4RepCT, spontaneously forms macroscopic fibers that resemble native spider silk.1,5 These are the first synthetic spider fibers reported to be formed in vitro in a physiological buffer without using toxic additives, which improves their potential in the area of biomaterial sciences. However, since 4RepCT is produced recombinantly in a bacterial host, all traces of the host must be removed before usage in vivo. Due to cost-efficiency, flexibility and suitability for large scale cultivation, the gram-negative bacterium Escherichia coli is nowadays a common host for recombinant protein production. Lipopolysaccharide (LPS), an integral component of the outer membrane of gram-negative bacteria, is endotoxic (pyrogenic) and can activate the innate immune system even at low concentrations. Conventional affinity purification of recombinantly produced proteins usually yields proteins with over 90% purity, but a significant amount of endotoxins is retained. Many different processes have been used for removal of LPS, for example, two-phase extraction, ultrafiltration, membrane adsorbers, and various LPS-selective affinity resins (reviewed in6-8). Many of these processes rely on more or less specific molecular properties of LPS, and the * To whom correspondence should be addressed. Tel.: +46-18-4714191. Fax: +46-18-550762. E-mail: [email protected]. † Swedish University of Agricultural Sciences. ‡ University of Konstanz. § Oslo University.

separation of LPS from protein is thus dependent on the properties of the protein of interest. Recently, highly LPSselective affinity matrices, called EndoTrap Blue and Red, with ligands based on selected affinity proteins, have been developed.9 Bacterial hosts for recombinant protein production are far from the only source of endotoxins, because bacteria may grow under various conditions, including nutrient poor water. In fact, endotoxin contamination is hard to avoid in most laboratory environments where biomaterials are synthesized, which complicates studies of biocompatibility.10 The most common route for detection of bacterial endotoxin is through Limulus amoebocyte lysate (LAL) assays, based on a cell lysate of the horseshoe crab Limulus polyphemus that coagulates in the presence of LPS. One drawback of LAL assays is that they are not suitable for solid samples; only liquid extracts can be measured. Recently, an in vitro pyrogen test (IPT),11,12 a new method for measurements of pyrogenicity that depends on cytokine release of human whole blood, was validated.13 The IPT method is suitable also for direct measurements of pyrogenic contamination of solid samples, such as protein fibers. Another advantage is that the IPT gives a value of the total pyrogenicity of the sample, that is, including the effect of the material itself and other contaminants that are pyrogenic in humans, and not only the LPS content. Herein, a successful production route of recombinant spider silk fibers of low pyrogenicity, measured by the IPT method, is described and its implications for cell culture are analyzed. The morphology, structure, and mechanical properties of the fibers are retained after autoclaving, and the fibers support growth of human primary fibroblasts. Moreover, fibers produced using this method have recently been used for analysis of in vivo biocompatibility by subcutaneous implantation in rats.14

10.1021/bm9014039  2010 American Chemical Society Published on Web 03/17/2010

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Biomacromolecules, Vol. 11, No. 4, 2010

Figure 1. LPS-depleting cell wash procedure. LPS is represented by black dashes.

Experimental Section Protein Expression. The miniature spider silk protein 4RepCT was expressed in E. coli BL21(DE3) cells (Merck Biosciences) using a modified pET32 vector encoding the fusion protein thioredoxin/His6tag/S-tag/thrombin cleavage site/4RepCT, as previously described.1 The cells were grown at 30 °C in ampicillin supplemented Luria-Bertani medium to an OD600 of ∼1, induced with 0.5 mM isopropyl-β-Dthiogalactopyranoside, and further incubated for 4 h at room temperature. LPS-Depleting Cell Wash Procedure. Cells were harvested by centrifugation at 4000 × g and gently resuspended in 60 mL wash buffer (100 mM Tris-HCl, pH 8.0) per initial liter of cell culture. Thereafter, the cells were subjected to a washing procedure, as described in Figure 1, through repeated centrifugation (4000 × g for 8 min at 16 °C) and gentle resuspension in wash buffer supplemented with (1) 5 mM CaCl2, (2) 10 mM EDTA, and (3) no supplements. After a final centrifugation step, the cells were resuspended in one of the following purification buffers: (A) 0.5 M NaCl, 20 mM Tris, pH 8, (B) 20% glycerol, 20 mM Tris, pH 8, or (C) 20 mM Tris, pH 8, and frozen at -20 °C. Protein Purification and Fiber Formation. After complete cell lysis with lysozyme and DNaseI, the 15000 × g supernatants were loaded on columns packed with Ni-sepharose (GE Healthcare, Uppsala, Sweden) and equilibrated with the corresponding purification buffer. The columns were washed extensively (20 column volumes (CV)) before bound proteins were eluted with 100 mM imidazole. For purification with buffer C (20 mM Tris only) an extra column wash step (5 CV) using either 0.1% w/v Triton X-114 at 4 °C, 20% aqueous isopropanol or 20% aqueous 1,2-hexandiol was tried. This was followed by an extra wash step using 10 CV of Buffer C to remove the used additives. Pooled fractions were dialyzed against 20 mM Tris-HCl, pH 8.0 overnight. Before loading onto EndoTrap Blue columns (Profos AG, Regensburg, Germany) 100 µM CaCl2 was added to the protein samples. After the void volume was discarded, the flow through was collected and diluted to a concentration of 1 mg/mL. 4RepCT was released from the tags by proteolytic cleavage using a thrombin/fusion protein ratio of 1:1000 (w/w) and allowed to self-assemble into fibers as previously described.1 Protein samples were separated on SDS-PAGE gels and then stained with Coomassie Brilliant Blue R-250. Protein content was determined from absorbance at 280 nm. Fiber Wash Procedure and Sterilization. Fibers were removed from the tubes and washed twice in 20 mM Tris buffer and twice in sterile water to remove any traces of the soluble released tags. The fibers were autoclaved for 15 min at 121 °C and 2.8 bar in tubes filled with sterile water. Pyrogen Analysis. The pyrogen content in protein solutions and fibers was measured using the IPT.11,12 Briefly, human whole blood was brought into direct contact with the samples (solutions or fibers) and the release of the pro-inflammatory cytokine IL-1β was measured. LPS concentration-response curves were done using LPS from

Hedhammar et al. Salmonella abortus equi. To exclude interference, parallel samples were spiked with a known concentration of LPS. Spike retrieval was considered successful if the signal corresponded to 50-200% of the signal achieved with the same concentration of LPS in the absence of a sample. For comparison, some of the soluble protein samples were also measured using a traditional LAL assay test kit (Associates of Cape Cod inc., Falmouth, MA). Thermogravimetric Analysis (TGA). Dried fibers were analyzed by TGA (Q500, TA Instruments). Samples were heated from 25 to 500 °C at a heating rate of 10 °C/min while the weight loss was measured. Dynamic Mechanical Analysis (DMA). To evaluate the impact of autoclave treatment on the mechanical performance of the fibers, tensile testing was carried out using a Q800 dynamic mechanical analyzer (TA Instruments) equipped with a tension clamp. Untreated or autoclaved fiber bundles (Ø ∼ 100 µm) were mounted in an adjustable holder, carefully straightened, and air-dried for approximately 20 min. The dried fiber bundles were glued to a double paper frame with a distance of 1.0 cm between attachment points. The frame was subsequently mounted in the tensile cell, taking care not to fix the clamps over the fibers. All measurements were carried out by applying a constant load of 0.2 N/min until sample failure. Scanning Electron Microscopy (SEM). Fibers, before and after autoclaving, were applied to SEM stubs and air-dried overnight. The samples were vacuum-coated with a thin layer of gold and palladium. Specimens were observed and photographed with a LEO 1550 FEG microscope (Carl Zeiss, Oberkochen, Germany) using an acceleration voltage of 10 kV. Structural Characterization. Circular dichroism (CD) spectroscopy was used to determine the secondary structure content of fibers before and after autoclaving. The fibers were cut in small pieces and suspended by vigorous vortexing in 2% SDS in water. The fibers were not dissolved by this treatment, but only suspended to small enough particles to allow CD measurments. CD spectra from 250 to 190 nm were recorded at 22 °C in a 0.1 cm path length quartz cuvette using a J-810 spectropolarimeter (Jasco). X-ray diffraction of bundles consisting of five aligned and dried fibers (before and after autoclaving, respectively) was collected on an inhouse Cu KR (λ ) 1.5418 Å) rotating anode source using a MarCCD345 detector. Data were collected for 60 min and sequential background measurements were subtracted. Chemical Stability. To investigate chemical stability, fibers were incubated in water, 1×PBS, 8 M urea, or 6 M guanidine hydrochloride (GdnHCl), respectively, for up to one week. The fibers were visually inspected, and in order to detect released protein, the supernatants were analyzed using SDS-PAGE. Fibroblast Culture. Human neonatal dermal fibroblasts (European Collection of Cell Cultures, P8) were used for culture on fibers. Fibroblasts were maintained in Dulbeccos Modified Eagle Medium supplemented with 5% fetal bovine serum, penicillin G (60 mg/L), streptomycin sulfate (50 mg/L), and 5% CO2 at 37 °C. Medium was changed every two to three days. Fibers used for cell culturing were sterilized by autoclave treatment in distilled H2O that was subsequently replaced by sterile PBS, pH 7.4, and the fibers were left to equilibrate for 72 h. To adhere to the substrate, fibers were placed on cell culture chamber slides (LabTek II) and allowed to air-dry in a laminar flow hood overnight. Prior to cell seeding, the dry fibers were incubated with 200 µL of complete growth medium at 37 °C for 1 h. Fibroblasts were seeded at 20000 cells/cm2 and allowed to grow for up to 7 days under the culture conditions specified above. Following 8 h, 24 h, and 7 days in culture, cells were stained with LIVE/DEAD fluorescent staining (Invitrogen, OR), according to the manufacturer’s recommendations, and imaged using a Nikon TE2000-U inverted microscope equipped with a high power mercury lamp.

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Table 1. Pyrogen Content of IMAC Purified Fusion Protein Recovered with and without a Cell Wash Procedure LPS content [µg LPS/mg recovered protein]a mean value of three parallel samples ( s.d.

protein recovery [mg/L cell culture OD600 ∼ 1] mean value of three parallel samples ( s.d.

1 ( 0.1 0.1 ( 0

42 ( 1 40 ( 1

without cell washes after cell washes a

125 pg of LPS from Salmonella abortus equi corresponds to 1 EU.

Table 2. Pyrogen Content in the Eluate After IMAC Purification Using Three Different Buffers

0.5 M NaCl 20% glycerol 20 mM Tris, pH 8 a

number of samples

LPS level [×104 pg/mg recovered protein]a mean value ( s.d.

5 5 5

14 ( 2 15 ( 2 10 ( 2


Results A miniature spider silk protein, 4RepCT, was recombinantly produced as a fusion protein in E. coli. To obtain fibers with low pyrogenic content, several different purification steps were tested. LPS-Depleting Cell Wash Procedure Decreases the LPS Content by 90%. Before disrupting the E. coli cells, which releases the intracellularly produced target protein, a cell wash procedure was included, as described in Figure 1. First, the cells were washed with 100 mM Tris buffer to remove LPS molecules that had been released into the media during cell growth. Then the cells were washed with a buffer containing Ca2+ ions, which are suggested to link LPS monomers by acting as bridges between phosphate groups.15,16 The next wash step includes twice as high concentration of EDTA as the Ca2+ concentration used in the previous step, in order to capture Ca2+ and other divalent cations that can link LPS molecules. Thereafter, the cells were washed in 100 mM Tris buffer to wash away the LPS molecules that were released from the outer membrane by the Ca2+/EDTA wash. Finally, the cells were resuspended in 20 mM Tris buffer, disrupted using lysozyme, and the His6tagged fusion protein was purified using immobilized metal ion affinity chromatography (IMAC). The LPS content in the pooled IMAC eluate, normalized to the amount of pure target protein obtained, is shown in Table 1. Fractions from washed cell cultures contained about 10-fold lower levels of LPS (Table 1). The amount of target protein recovered after cell washes was only a few percentages lower than what was recovered without cell washes. Buffer Additives Do Not Reduce the LPS Content. After cell disruption, the target protein and the LPS molecules from the outer membrane will interact to different degrees, depending on the composition of the buffer used. Three different running buffers, based on 20 mM Tris buffer, pH 8, were evaluated: one containing 0.5 M of NaCl, one containing 20% glycerol, and one containing no additives. Purification in 20 mM Tris buffer without additives gave the lowest level of LPS (Table 2). Evaluation of the SDS-PAGE results of purified fractions with QuantityOne software revealed that purification in 20 mM Tris buffer gave a fusion protein of 95% purity, while a lower purity (85%) was obtained through purification in the same buffer supplemented with 0.5 M NaCl (data not shown). Inclusion of 20% glycerol in the buffer did not seem to have an impact on protein purity. While the fusion protein is captured on the IMAC resin it is possible to introduce a column wash step. One detergent and two solvents were evaluated as additives to 20 mM Tris buffer:

0.1% Triton X114 at 4 °C, 20% of isopropanol, and 20% of 1,2-hexandiol. Neither of these buffers were found to decrease the pyrogenic activity in the eluated protein to any substantial extent (data not shown). EndoTrap Columns Reduce LPS Content by a Factor of 103. After dialysis against 20 mM Tris buffer, pH 8, 100 µM CaCl2 was added to the IMAC purified fusion protein solution, which was then passed through EndoTrap Blue columns equilibrated with the same buffer. This step reduced the LPS content by a factor of 103 without any significant protein loss (Table 3). For comparison, EndoTrap Red columns were used, which gave the same results (data not shown). LPS-Depleted 4RepCT Allow Fiber Formation. The recombinant spider silk protein 4RepCT was allowed to selfassemble into fibers (Figure 2) when released from the tags by proteolytic cleavage. The formed fibers were washed with 20 mM Tris buffer and sterile water to remove any traces of soluble protein, mainly the released tags. The pyrogen content in the fibers was measured using IPT and was found to be lower than in the protein solution from which they were formed (Table 3). Sterilization by Autoclave Treatment Does Not Significantly Affect the Fibers. To sterilize the fibers they were autoclaved in sterile water. According to visual inspection and SEM pictures, collected before and after autoclaving, the fiber morphology was not affected by this treatment (Figure 2). However, some of the fibers were loosely entangled after autoclaving, which can also happen if the fibers are shaken in water. To determine whether autoclave treatment had any effect on the mechanical performance, tensile testing of untreated and autoclaved fiber bundles was carried out. Most fiber bundles, irrespective of treatment, broke at the grip point during tensile testing. This may be caused by defects introduced by fixation or as a consequence of the fibers being difficult to align perfectly with the direction of pulling. Alignment was especially problematic for those fibers that were entangled during autoclaving, hence, the lower amount of autoclaved fibers analyzed. A majority of the samples, both control and autoclaved fibers, broke at the grip point during tensile testing. The maximum stress and maximum strain could therefore not be reliably estimated using this set up. Therefore, only the modulus of the tensile curve was considered herein. No significant difference in the tensile modulus was observed between untreated and autoclaved fibers (Table 4). In support of these results, the thermogravimetric analysis of dried fibers (Figure 3) showed an onset point of thermal degradation (defined as the intercept between the initial slope and the maximum slope of the degradation phase) at 267 °C, that is, well above the temperature used for autoclaving. The fibers were shown to not dissolve during long-term incubation in water or denaturing agents such as urea and GdnHCl. However, fibers could be dissolved in neat formic acid supplemented with SDS. Subsequent SDS-PAGE analyses showed no degradation products of autoclaved or untreated fibers (data not shown). CD spectra of suspended fibers showed a single minimum around 220 nm and a maximum at 193, thus indicating a substantial fraction of β-sheet structure. No difference could

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Table 3. Pyrogenicity and Recovery of Fusion Protein After Different Purification Steps number of samples

LPS level [pg/mg protein]a mean value ( s.d.

6 6 9

1 × 105 ( 1 × 104 320 ( 150 40 ( 40

IMAC eluate EndoTrap eluate fiber a

protein recovery [mg/L cell culture OD600 ∼ 1] mean value of three parallel samples ( s.d. 40 ( 1 39 ( 1 not determined


Figure 2. Morphology of the fibers. Photographs (A, B) and SEM micrograhs (C, D) of fibers before (A, C) and after (B, D) sterilization through autoclaving. Table 4. Tensile Modules of the Fibers of Control and Autoclaved Fibers

control autoclaved

number of samples

modulus [GPa] mean value ( s.d.

11 6

6(1 6(2

be seen after autoclaving of the fibers (Figure 4A). This is coherent with the X-ray diffraction patterns of autoclaved fibers that show up on a similar β-sheet structure as before autoclaving (Figure 4B,C). Fibers Support Adherence and Growth of Fibroblasts. Human dermal fibroblasts were cultured in the presence of fibers for up to 7 days and investigated with the LIVE/DEAD viability assay at different time points (Figure 5). The green fluorescence stems from calceinAM, which is cleaved to fluorescent calcein by live cells. The nuclei of dead cells are stained red by the uptake of ethidium homodimer through compromised cell membranes and the subsequent staining of polynucleic acids. This agent also stains the fibers red, which makes this method

Figure 4. Structural characterization of the fibers. (A) CD spectra of untreated (thick line) and autoclaved (thin line) fibers suspended in 2% SDS. (B) X-ray diffraction pattern of untreated (left half) and autoclaved (right half) fibers. (C) Intensity as a function of scattering angle 2θ of untreated (thick line) and autoclaved (thin line) fibers.

Figure 3. Thermal stability of the fibers. Thermogravimetric analysis of 4RepCT fibers from 25 to 500 °C.

very effective for viewing live cell-fiber interactions. It is still possible to detect dead cells on the fibers, although it can be difficult to distinguish them from overlapping fibers. However, it is clear from Figure 5 that fibroblasts adhere to and grow directly adjacent to fibers already 8 h post-seeding. With the higher cell density observed at day 7, cells can be seen aligning with the fibers and also covering them to a higher degree.

Sterilized Spider Silk of Low Pyrogenicity

Figure 5. Fibroblasts grown on the fibers. Live (green)/Dead (red) staining of human dermal fibroblasts after 8 h and 7 days of culture in the absence and presence of fibers. The original magnification is noted above each column.

Discussion A recombinant miniature spider protein, 4RepCT, can be produced at comparatively high levels in soluble form when fused to a soluble protein partner.1,5 In this way, the need for denaturing or chaotropic agents for recovery of recombinant spider silk protein is avoided. Moreover, in contrast to other existing processes for formation of artificial protein fibers, the natively purified 4RepCT spontaneously forms fibers in a physiological buffer, without the need for spinning into coagulation baths containing harsh solvents. Fibers can thus be produced under physiological-like conditions throughout the process. However, as 4RepCT is produced in a gram-negative host, bacterial endotoxins have to be thoroughly removed before in vivo use of the fibers. Ideally, the intracellularly produced protein and LPS molecules from the outer bacterial membrane would never meet. Unfortunately this is unavoidable, but the LPS level before cell lysis can quite easily be reduced, using a simple cell wash procedure (Figure 1, Table 1). LPS molecules are continuously liberated from the bacterial cell wall into the medium, during growth and division, as well as upon cell death.7,17 LPS molecules released into the growth medium during cultivation can easily be removed by washing the cells before disrupting them. In 1965, Leive discovered that up to 50% of the LPS molecules could be released from the cell wall of E. coli by the addition of EDTA.18 This is probably due to the ability of EDTA to capture divalent cations that link LPS molecules via their phosphate groups. However, higher concentrations of EDTA or longer incubation times did not increase the release, suggesting that several fractions of LPS exist in the membrane.19 The EDTA-insensitive fraction was later suggested to contain newly synthesized LPS molecules.20 Preincubation of cells with Ca2+ prior to EDTA treatment was found to somewhat increase the portion of released LPS, while less protein and lipids were extracted.19,21 The mechanism behind this phenomenon is not revealed yet, but the increased Ca2+ concentration might shift LPS molecules into a certain conformation that is sensitive to EDTA treatment. The LPS deleting cell wash procedure used in the present study, including an initial cell wash and subsequent Ca2+ and EDTA treatments, reduces the LPS content in the fraction of IMAC purified fusion protein by up to 90% (Table 1). The reduction gained by the cell wash procedure might be further increased during the IMAC purification, because a lower initial LPS concentration disfavors LPS-protein interactions. The amount of protein recovered is only minimally affected by the cell wash procedure (Table 1), which indicates that the cell walls are disrupted to a marginal extent, despite the fact that several centrifugation steps are introduced. A cell wash procedure, as outlined here, is simple to perform and of low cost, thus, it is worth including before using more expensive purification steps.


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The amphiphilic nature of LPS, due to phosphorylated oligosaccharides and fatty acyl chains, enables both electrostatic and hydrophobic associations with the target protein, which cause most proteins to bind LPS, although to different degrees. The strength of the protein-LPS interactions is affected by the environment; therefore, the choice of lysis and purification buffer is of importance. Also, 4RepCT has an amphiphilic nature, due to polyalanine blocks and more polar regions, and can provide sites for both hydrophobic and charge interactions. The amounts of LPS copurified with 4RepCT are increased both in high salt and in glycerol buffer compared to low conductivity buffers (Table 2), which implies that the 4RepCT-LPS interactions are both electrostatic and hydrophobic. Different detergents (e.g., Triton X-11422) and organic solvents (e.g., isopropanol,23 1,2-alkanediols24) have previously been employed to improve LPS clearance. The capture of a fusion protein on an affinity matrix provides the opportunity to subject the protein to different washing procedures. Column washes with either Triton X-114, isopropanol, or 1,2-hexandiol did not decrease the pyrogenicity of the eluted protein fractions (data not shown). However, isopropanol was used at a 3-fold lower concentration than previously suggested,23 because it otherwise increased the aggregation of the fusion protein (data not shown). From these results we concluded not to use such additives because they may affect the protein and also have to be removed completely before in vivo use of the fibers. By the same argumentation, LPS removal using, for example, resins coupled with polymyxin B, which is toxic,25 was avoided. The comparatively high cost of the EndoTrap matrices requires an inexpensive initial purification step to render a costeffective process. Therefore, these columns were used only after an initial LPS reduction through cell washes and after IMAC purifications, which provides a concentrated target protein in a small volume. Passage though an EndoTrap Blue column led to a LPS reduction by up to a factor of 103, which is somewhat higher than specified by the supplier (www.profos.de). Recombinant production of 4RepCT in fusion with the solubility enhancing thioredoxin tag does not only provide the protein in soluble form, but also results in increased stability of the protein, which allows the multiple purification steps needed for removal of LPS. After passage through the LPS depleting EndoTrap column, the pyrogenicity of the fusion protein is below 500 pg/mg protein (Table 3). The fusion protein is then proteolytically cleaved and the 4RepCT part is allowed to self-assemble into fibers. Only 5-20% of the LPS content in the cleavage mixture is trapped in the fiber, while approximately 70% is retained in the soluble supernatant after fiber formation, where the fusion tags are found (data not shown and Table 3). Whether LPS interacts more tightly with the tag than with 4RepCT is hard to tell. Analysis of fractions containing 4RepCT and Thioredoxin/His6/S-tag, respectively, separated, while both in soluble form by passage through a second IMAC column, gave similar pyrogenicity levels, which indicates that both parts interact equally with LPS (data not shown). The structural change of 4RepCT during fiber formation might affect LPS binding, thus, LPS molecules might be excluded from the fiber. Washing and autoclaving of the fibers might also reduce their pyrogenicity. It is also possible that LPS molecules are captured inside the fiber and thereby not detected by IPT. Solution samples that were measured using both the IPT and the LAL assay showed up on similar levels (data not shown), indicating that LPS is the major source of pyrogenic compounds in these samples. Regarding the solid fiber samples, it is possible, although not likely, that the pyrogenicity measured by IPT is

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partly due to an inherent pyrogenicity of the protein in fibrous form, as the IPT method measures the total cytokine release, irrespective of the triggering factor. Unfortunately, the direct LAL assay, measuring the actual LPS content, cannot be used for measurements of the fibers. Anyhow, the pyrogenicity of the fibers, whether entirely due to LPS or not, corresponds to only 5-100 pg LPS/mg, which converts to approximately 0.04-0.8 endotoxin units (EU)/mg. The U.S. Food and Drug Administration and the European Pharmacopoeias recommend that no more than 5 EU per kilogram of body weight should be introduced parenterally into a human or animal.8,26 The limit for medical devices is set to 20 EU.27 The achieved low pyrogenicity of the fibers thus allows implantation, which has encouraged us to further evaluate them for usage as a biomaterial. The low pyrogenicity of the fibres was further confirmed when they were shown to be well accepted after subcutaneous implantation in rats.14 For practical reasons, a biomaterial must be easily sterilized. Autoclave treatment is a preferred sterilization method, although it is not compatible with several biomaterials, for example, collagen28 and polyurethanes.29 Native silks have been shown to be thermally stable.30,31 TGA of dried 4RepCT-fibers showed an onset point of degradation at 267 °C. This temperature is well above the autoclave temperature (121 °C). However, because autoclaving is performed in water (neutral), it can not be completely ruled out that hydrolysis of some susceptible peptide bonds can occur, but SDS-PAGE of dissolved fibers does not show detectable degradation products. The tensile modulus, as well as nanometre scale morphology, are also unaffected after autoclaving (Table 4 and Figure 2). CD and X-ray diffraction show that the predominantly β-sheet structure of the fiber is unaffected by autoclaving. Further complementary studies, such as infrared or Raman spectroscopy, may possibly resolve minor structural changes, if they exist. Together, these results indicate that autoclave treatment may be recommended for sterilization of these fibers. Sterilization by submersion in ethanol did not affect the structure to any substantial extent either, as judged by a similar X-ray diffraction pattern (data not shown). The chemical stability of the fibers was further confirmed by the fact that submersion in 8 M urea or 6 M GdnHCl for 7 days did not release any protein (data not shown). When implanted in vivo, the fibers are eventually degraded, possibly by endocytosis and subsequent intracellular proteolysis by macrophages.14 To further evaluate 4RepCT-fibers for usage as biomaterial, its compatibility for cell culturing was investigated. This showed that the fibers are not harmful to human primary fibroblasts, which adhered to and grow directly adjacent to fibers. After 7 days of culture, cells aligned with the fibers and also covered them to a higher degree (Figure 5). Moreover, the fibers support ingrowth of fibroblasts and newly formed capillaries when implanted subcutaneously in rats.14

Conclusions We demonstrate a method for production of a synthetic spider silk with documented low pyrogenicity. The procedure for LPS removal now presented is reproducible and easy to perform. Moreover, the fibers are nondestructively sterilized by autoclave treatment and allow growth of human primary fibroblasts. We believe that the recombinant spider silk fibers are of particular interest for biomaterial science. Acknowledgment. We thank Bjo¨rn Atthoff for help with scanning electron microscopy and Marie Svensson for help with

Hedhammar et al.

the LAL assay. This study was supported by a European Commission grant (“Spiderman” Contract No. G5RD-CT-200200738), the Swedish Research Council, Formas and Vinnova.

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Sterilized Recombinant Spider Silk Fibers of Low Pyrogenicity

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