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Recombinant Silk Production in Bacteria
Recombinant Silk Production in Bacteria$ DL Kaplan, Tufts University, Medford, MA, United States T Scheibel, University of Bayreuth, Bayreuth, Germany r 2017 Elsevier Inc. All rights reserved.
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Introduction Silk Processing Properties of Silks Silk Genetics and Cloning Future Directions
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Introduction
Silks can be defined as secreted proteins spun into fibrous structures. Characteristically, silk proteins are produced in specialized glands and stored in a fluid state in the lumen of the gland. As the fluid passes through the spinning duct, a rapid transformation to the solid state takes place and the silk becomes water-insoluble. Silk fibers are diverse in function, depending on the biological source, such as spiders or silkworms. Silkworm silks form protective coatings for developing larvae, while spider silks function in prey capture, reproduction, vibrational sensors, safety lines, and dispersion tools. A limited number of silkworm and spider silks have been characterized and they generally consist of glycine-enriched sequences that assemble into less crystalline regions of the materials, and poly(alanine) or poly(glycine–alanine) repeats that form b-sheet crystalline structures. For recent reviews on silk structures see Heim et al. (2009), Heidebrecht and Scheibel (2013), and Eisoldt et al. (2012). Reeled silkworm silk (Bombyx mori) has been used extensively in the textile industry for over 5000 years. Sericin is extracted from the cocoons by immersion in hot soapy water, leaving 300 to 1200 m of usable fiber. Unlike silkworm silks, spider silk production has not been domesticated for textile material applications. This is because spiders are more difficult to raise in large numbers, owing to their solitary and predatory nature, and because orb webs which contain up to five different types of silk are not reelable as a single fiber like the cocoon silk from the silkworm. Further, in comparison to the silkworm, spiders generate only small quantities of silk. Despite the intriguing mechanical features of spider silks, the difficulty in domesticating spiders necessitates an alternative approach to silk production: genetic engineering and recombinant production.
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Silk Processing
The processing of water-soluble high molecular weight silk proteins into water-insoluble fibers involves many factors, including acidification, salting out/phase separation, and perhaps other chemical or physical steps. The vast majority of silk proteins is large and consists of a core repetitive region, being made up of sequence motifs that can be repeated several hundred times (Guerette et al., 1996; Hayashi and Lewis, 1998, 2000). This core region imparts the physical properties onto the resulting silk thread. Most of the proteins then have non-repetitive terminal sequences with a unique fold in the protein. In the case of Major Ampullate spider silk proteins it has been shown that the termini act as a switch being involved in the storage of the proteins and triggering the protein to convert from a soluble to an insoluble fiber with aligned crystalline b-sheet regions (Eisoldt et al., 2012). In the spinning duct, changes in physiological conditions such as pH and salt concentrations accompany and further trigger the structural conversion/assembly, and physical shear generated during spinning finalizes silk fiber processing (Jin and Kaplan, 2003; Heim et al., 2009; Eisoldt et al., 2011). The highly organized fibrous structure of silk and the extensive hydrogen bonding and van der Waals interactions lead to the exclusion of water from the intersheet regions of the b-sheets after silk spinning. Most silk fibers are insoluble in water, dilute acids and alkali, chaotropic agents such as urea and guanidine hydrochloride, and most organic solvents. Silk can be solubilized by immersion of the fibers in very high concentration salt solutions such as lithium bromide, lithium thiocyanate, guanidinium thiocyanate or calcium chloride and other calcium salts. High concentrations of propionic acid/hydrochloric acid mixtures, hexafluoroisopropanol and formic acid can also be used to facilitate solubility. After solubilization, dialysis into other chaotropic agents, water or buffers can be carried out (Kaplan et al., 1994; Hardy et al., 2008). ☆
Change History: June 2016. T. Scheibel revised the article completely.
Reference Module in Materials Science and Materials Engineering
doi:10.1016/B978-0-12-803581-8.02274-8
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Overview of spider silk cDNAs expressed in different host organisms
Type of silk Protein Major ampullate MaSp1
MaSp1 (fibroin chimera) MaSp1 and MaSp2
MaSp1- and MaSp2-collagen fusion protein ADF3 ADF3 and ADF4
Flagelliform Flag Polyhedron-flag fusion protein Tubiliform (aka cylindriform) TuSp1 TuSp1 Pyriform PySp2 a
Molecular weight (kDa)
Spider
Host organism
References
22, 25 10–28
Euprosthenops sp. Euprosthenops australis
Mammalian cells (COS-1) Bacteria (Escherichia coli)
43 83 12 14 60–140 31–66 33–39 57–61 60–140 60 50–105 35–56
Nephila clavipes Nephila clavata N. clavipes Latrodectus hesperus N. clavipes N. clavipes N. clavipes N. clavipes Araneus diadematus A. diadematus A. diadematus A. diadematus
Bacteria (E. coli) Transgenic animals (Bombyx mori) Bacteria (E. coli) Bacteria (E. coli) Mammalian cells (MAC-T and BHK)a Transgenic animals (mice) Yeast (Pichia pastoris) Yeast (P. pastoris) Mammalian cells (MAC-T and BHKa) Transgenic animals (goats) Insect cells (Spodoptera frugiperda) Insect cells (S. frugiperda)
35–56
A. diadematus
Insect cells (Trichopulsia ni)
Grip et al. (2006) Askarieh et al. (2010), Hedhammar et al. (2008), and Stark et al. (2007) Arcidiacono et al. (1998) Wen et al. (2010) Sponner et al. (2005) Hagn et al. (2010, 2011) Lazaris et al. (2002) Xu et al. (2007) Teule et al. (2003) Teule et al. (2003) Lazaris et al. (2002) Karatzas et al. (1999) Ittah et al. (2006) Huemmerich et al. (2004b) and Vendrely and Scheibel (2007) Vendrely and Scheibel (2007)
28 61
Araneus ventricosus A. ventricosus
Insect cells (S. frugiperda) Insect cells (S. frugiperda)
Lee et al. (2007) Lee et al. (2007)
12–15 33, 45
Nephila antipodiana L. hesperus
Bacteria (E. coli) Bacteria (E. coli)
Lin et al. (2009) Gnesa et al. (2012)
N/Ab
L. hesperus
Bacteria (E. coli)
Geurts et al. (2010)
MAC-T: bovine mammary ephithelial alveolar cells, BHK: baby hamster kidney cells. N/A: not applicable.
b
Recombinant Silk Production in Bacteria
Table 1
Recombinant Silk Production in Bacteria
3
3
Properties of Silks
The mechanical properties of silks include an intriguing combination of high strength and elasticity yielding an outstanding toughness (Cunniff et al., 1994; Xu and Lewis, 1990; Heidebrecht et al., 2015). Some spider silks exhibit over 200% elongation and others maintain tensile strengths approaching those of high performance fibers such as Kevlar. In terms of energy absorption prior to break, spider silks are unmatched in the world of synthetic or natural fibers. These properties are significantly higher for spider dragline silk than silkworm silks. The mechanical properties of silk fibers are a direct result of the size and orientation of the b-sheet (crystalline) domains, the connectivity of these domains to the amorphous domains, and the interfaces or transitions between amorphous and crystalline domains. The superior performance of spider silk in webs is, however, not due merely to its exceptional ultimate strength and strain, but arises also from the nonlinear response of silk threads to strain and their geometrical arrangement in a web (Cranford et al., 2012). Resistance to axial compressive deformation is another unique feature of these fibers observed with microscopic evaluations of knotted single fibers. No evidence of kink-band failure on the compressive side of knot curves was observed. Synthetic high performance fibers fail by this mode even at relatively low stress levels; this is a major limitation with synthetic fibers in many applications. In addition, spider silk shows a torsional shape memory that prevents the spider from twisting and turning during its descent on a silk thread (Emile et al., 2006). Interestingly, the silk fiber needs no extra stimulus for total recovery after being turned from its initial position. Instead, it scarcely oscillates after twisting because of its high damping coefficient. Spider silk also shows supercontraction (Perez-Rigueiro et al., 2003; Liu et al., 2005). Absorption of water leads to shrinkage and tightens the thread. This process is important to ensure the rigidity of the spider’s web during its lifetime and is thought to be caused by the organization and arrangement of individual silk proteins. Finally, spider dragline silk is thermally stable to about 2301C. Table 2
Overview of designed spider silk genes expressed in bacterial hosts
Type of silk Protein
Molecular weight (kDa)
Spider
Host organism
References
Major ampullate MaSp1
N/Aa 100–285 15–26
Latrodectus hesperus Nephila clavipes N. clavipes
Salmonella typhimurium Escherichia colib E. coli
45–60
N. clavipes
E. coli
MaSp1 (Gly-rich repeats) MaSp2
10–20 63–71
N. clavipes Argiope aurantia
E. coli E. coli
MaSp2/Flag MaSp1 and MaSp2
31–112 58, 62 20–56
N/A N. clavipes N. clavipes
E. coli E. coli E. coli
ADF3, ADF4
N/A 55, 67 15–41 65–163 34–134
Bacillus subtilis E. coli E. coli E. coli E. coli
ADF1, ADF2, ADF3, ADF4
25–56
N. clavipes N. clavipes N. clavipes N. clavipes Araneus diadematus A. diadematus
S. typhimurium
Widmaier et al. (2009) and Widmaier and Voigt (2010) Xia et al. (2010) Szela et al. (2000) and Winkler et al. (1999, 2000) Bini et al. (2006), Huang et al. (2007), and Wong Po Foo et al. (2006) Fukushima (1998) Brooks et al. (2008b) and Teule et al. (2009) Lewis et al. (1996) Teule et al. (2007) Arcidiacono et al. (2002) and Mello et al. (2004) Fahnestock (1994) Brooks et al. (2008a) Prince et al. (1995) Fahnestock and Irwin (1997) Huemmerich et al. (2004a) and Heidebrecht et al. (2015) Widmaier et al. (2009) and Widmaier and Voigt (2010)
N/A
N. clavipes
S. typhimurium
Flag
14–94
N. clavipes
E. coli
Flag (Gly-rich repeats)
25
N. clavipes
E. coli
Widmaier et al. (2009) and Widmaier and Voigt (2010) Vendrely et al. (2008) and Heim et al. (2010) Zhou et al. (2001)
90
Nephila antipodiana
E. coli
Oster et al. (2014)
76–89 18–36
N/A N/A
E. coli E. coli
Cappello et al. (1990) Yang and Asakura (2005)
Flagelliform silk Flag
Tubuliform/cylindriform silk TuSp1-GFP N/A N/A Gly-rich repeats and Alablocks a
N/A: not applicable. Metabolically engineered.
b
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Recombinant Silk Production in Bacteria
Silk Genetics and Cloning
Recently, remarkable progress has been made in understanding silk genetics, structures and biophysics, as well as their recombinant production. Biotechnological strategies concerning silk protein production have to consider several issues such as the size of the underlying silk genes and their highly repetitive sequences. Nowadays, cloning and expression of native silk sequences (cDNA) have been achieved in a variety of host systems as summarized exemplarily for spider silk in Table 1. The use of bacterial hosts, such as Escherichia coli, typically used in industrial processes, for the production of full-length native silk proteins is limited due to a distinct codon usage different to that of spiders and due to the size-limited yield of recombinant proteins in E. coli. Furthermore, bacterial hosts often remove repetitive sequences by a mechanism of homologue recombination. Therefore, expression of (partial) silk cDNA is often ineffective in E. coli. Since the codon usage of eukaryotic organisms is more closely related to that of insects and spiders, eukaryotic cells reflect more suitable expression hosts. Successful expression of, for example, cDNA fragments from two spiders, Nephila clavipes and Araneus diadematus, was achieved in bovine mammary epithelial and hamster kidney cells, as well as in insect cells. The drawback of these systems is the lack of easy and cheap scalability to yield high amounts of silk proteins. Since bacteria are easy to handle and can be grown in industrial scales at low costs, a way was sought to overcome the described complications. Therefore, synthetic genes with optimized codon usage or variants based on consensus repeat sequences garnered from data from native genes, as well as variants combining consensus sequences with natural sequences, for example, encoding terminal domains have been engineered and expressed in bacteria (exemplarily shown for different spider silks in Table 2). The described protein engineering technique yields mimetics of spider silk as well as new proteins with additional features not found in natural silk proteins. For instance, it has been investigated whether the incorporation of specific amino acids influences protein solubility and allows triggering of silk assembly. To gain new functional properties, incorporation of amino acids with chemically active side chains, like lysine or cysteine, is also feasible, since this allows side specific functionalization of the silk proteins. Even the addition of oligopeptides is conceivable. In this context chimeric proteins consisting of a spider silk domain and a peptide known to precipitate silica or the cell recognition peptide RGD have been successfully engineered. Thus, chimeric silk proteins can comprise chemically active sites, enzymatic activity, and receptor binding sites, among other functionalities (Humenik and Scheibel, 2014; Borkner et al., 2014; Schacht and Scheibel, 2014). Importantly, bacteria are not the only suitable scalable hosts expressing engineered recombinant silks, and therefore a variety of hosts including yeast, plants, insects and even goats has been tested as shown exemplarily for spider silk in Table 3.
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Future Directions
Novel and important applications for silk proteins can be envisioned, owing, for example, to the unique mechanical properties of the fibers made thereof (Heidebrecht et al., 2015). Further, plenty of studies have been focused on silk-based biomaterials, due to Table 3
Overview of designed spider silk genes expressed in different eukaryotic hosts
Type of silk Protein Major ampullate MaSp1
MaSp2
MaSp1 and MaSp2 Flagelliform Flag N/A Amphiphilic silk-like protein a
N/A: not applicable.
Molecular weight (kDa)
Spider
Host organism
References
N/Aa 94
Nephila clavipes N. clavipes
Yeast (Pichia pastoris) Yeast (P. pastoris)
64, 127
N. clavipes
Plants (Arabidopsis thaliana)
13–100
N. clavipes
Plants (Nicotiana tobaccum)
13–100
N. clavipes
Plants (Solanum tuberosum)
70 70 113
Insect cells (Bombyx mori) Transgenic animals (B. mori) Yeast (P. pastoris)
65 60 50
Nephila clavata N. clavata Nephila madagascariensis N. clavipes N. clavipes N. clavipes
Fahnestock and Bedzyk (1997) Agapov et al. (2009) and Bogush et al. (2009) Barr et al. (2004) and Yang et al. (2005) Scheller and Conrad (2005) and Scheller et al. (2001, 2004) Scheller and Conrad (2005) and Scheller et al. (2001, 2004) Zhang et al. (2008) Zhang et al. (2008) Bogush et al. (2009)
Plants (N. tobaccum) Plants (N. tobaccum) Transgenic animals (goat)
Patel et al. (2007) Menassa et al. (2004) Perez-Rigueiro et al. (2011)
37
N. clavipes
Insect cells (B. mori)
Miao et al. (2006)
28–32
N/A
Yeast (P. pastoris)
Werten et al. (2008)
Recombinant Silk Production in Bacteria
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the realization that silk proteins are non-immunogenic. The option to genetically tailor composition and structure provides a basis for new types of biomaterials based on silks for a variety of biomedical needs, including sutures, implant coatings, drug delivery systems, or scaffolds for tissue engineering (Leal-Egana and Scheibel, 2010; Schacht and Scheibel, 2014). Biofabrication is a new technology to simultaneously 3D-print materials together with cells in which silk proteins show extraordinary properties when used as bioinks (Schacht et al., 2015; DeSimone et al., 2015; Jüngst et al., 2016). However, also technical applications are envisioned in the near future such as filter devices, photonics, optical or electrical devices (Omenetto and Kaplan, 2010; Tao et al., 2012; Borkner et al., 2014).
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Recombinant Silk Production in Bacteria
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Ed. 54, 2816–2820. Schacht, K., Scheibel, T., 2014. Processing of recombinant spider silk proteins into tailor-made materials for biomaterials applications. Curr. Opin. Biotech. 29, 62–69. Scheller, J., Conrad, U., 2005. Plant-based material, protein and biodegradable plastic. Curr. Opin. Plant Biol. 8, 188–196. Scheller, J., Guhrs, K.H., Grosse, F., Conrad, U., 2001. Production of spider silk proteins in tobacco and potato. Nat. Biotech. 19, 573–577. Scheller, J., Henggeler, D., Viviani, A., Conrad, U., 2004. Purification of spider silk-elastin from transgenic plants and application for human chondrocyte proliferation. Transgenic Res. 13, 51–57. Sponner, A., Vater, W., Rommerskirch, W., et al., 2005. The conserved C-termini contribute to the properties of spider silk fibroins. Biochem. Biophys. Res. Com. 338, 897–902. Stark, M., Grip, S., Rising, A., et al., 2007. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. Biomacromolecules 8, 1695–1701. Szela, S., Avtges, P., Valluzzi, R., et al., 2000. Reduction–oxidation control of beta-sheet assembly in genetically engineered silk. Biomacromolecules 1, 534–542. Tao, H., Kaplan, D.L., Omenetto, F.G., 2012. Silk Materials – A road to sustainable high technology. Adv. Mater. 24 (21), 2824–2837. Teule, F., Aube, C., Ellison, M., Abbott, A., 2003. Biomimetic manufacturing of customised novel fibre proteins for specialised applications. AUTEX Res. J. 3, 160–165. Teule, F., Cooper, A.R., Furin, W.A., et al., 2009. A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nat. Prot. 4, 341–355. Teule, F., Furin, W.A., Cooper, A.R., Duncan, J.R., Lewis, R.V., 2007. Modifications of spider silk sequences in an attempt to control the mechanical properties of the synthetic fibers. J. Mater. Sci. 42, 8974–8985. Vendrely, C., Ackerschott, C., Romer, L., Scheibel, T., 2008. Molecular design of performance proteins with repetitive sequences: Recombinant flagelliform spider silk as basis for biomaterials. Methods Mol. Biol. 474, 3–14. Vendrely, C., Scheibel, T., 2007. Biotechnological production of spider silk proteins enables new applications. Macromol. Biosci. 7, 401–409. Wen, H.X., Lan, X.Q., Zhang, Y.S., et al., 2010. Transgenic silkworms (Bombyx mori) produce recombinant spider dragline silk in cocoons. Mol. Biol. Rep. 37, 1815–1821. Werten, M.W., Moers, A.P., Vong, T., et al., 2008. Biosynthesis of an amphiphilic silk-like polymer. Biomacromolecules 9, 1705–1711. Widmaier, D.M., Tullman-Ercek, D., Mirsky, E.A., et al., 2009. Engineering the Salmonella type III secretion system to export spider silk monomers. Mol. Syst. Biol. 5, 309. Widmaier, D.M., Voigt, C.A., 2010. Quantification of the physiochemical constraints on the export of spider silk proteins by Salmonella type III secretion. Microb. Cell Fact. 9, 78. Winkler, S., Szela, S., Avtges, P., et al., 1999. Designing recombinant spider silk proteins to control assembly. Int. J. Biol. Macromol. 24, 265–270. Winkler, S., Wilson, D., Kaplan, D.L., 2000. Enzymatic phosphorylation/dephosphorylation to control silk structure. Biochemistry 39, 12739–12746. Wong Po Foo, C., Patwardhan, S.V., Belton, D.J., et al., 2006. Novel nanocomposites from spider silk–silica fusion (chimeric) proteins. Proc. Nat. Acad. Sci. 103, 9428–9433. Xia, X.-X., Qian, Z.-G., Ki, C.S., et al., 2010. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc. Nat. Acad. Sci. 107, 14059–14063. Xu, H.T., Fan, B.L., Yu, S.Y., et al., 2007. Construct synthetic gene encoding artificial spider dragline silk protein and its expression in milk of transgenic mice. Anim. Biotech. 18, 1–12. Xu, M., Lewis, R.V., 1990. Structure of a protein superfiber: Spider dragline silk. Proc. Natl. Acad. Sci. USA 87, 7120–7124. Yang, J.J., Barr, L.A., Fahnestock, S.R., Liu, Z.B., 2005. High yield recombinant silk-like protein production in transgenic plants through protein targeting. Transgenic Res. 14, 313–324. Yang, M., Asakura, T., 2005. Design, expression and solid-state NMR characterization of silk-like materials constructed from sequences of spider silk, Samia cynthia ricini and Bombyx mori silk fibroins. J. Biochem. 137, 721–729. Zhang, Y., Hu, J., Miao, Y., et al., 2008. Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield. Mol. Biol. Rep. 35, 329–335. Zhou, Y.T., Wu, S.X., Conticello, V.P., 2001. Genetically directed synthesis and spectroscopic analysis of a protein polymer derived from a flagelliform silk sequence. Biomacromolecules 2, 111–125.
Further Reading Augsten, K., Muhlig, P., Herrmann, C., 2000. Glycoproteins and skin-core structure in Nephila clavipes spider silk observed by light and electron microscopy. Scanning 22, 12–15. Schmidt, F.R., 2004. Recombinant expression systems in the pharmaceutical industry. Appl. Microbiol. Biotechnol. 65, 363–372.
1 2 3 4 5 References Further Reading
1
Introduction Silk Processing Properties of Silks Silk Genetics and Cloning Future Directions
1 1 1 4 4 5 6
Introduction
Silks can be defined as secreted proteins spun into fibrous structures. Characteristically, silk proteins are produced in specialized glands and stored in a fluid state in the lumen of the gland. As the fluid passes through the spinning duct, a rapid transformation to the solid state takes place and the silk becomes water-insoluble. Silk fibers are diverse in function, depending on the biological source, such as spiders or silkworms. Silkworm silks form protective coatings for developing larvae, while spider silks function in prey capture, reproduction, vibrational sensors, safety lines, and dispersion tools. A limited number of silkworm and spider silks have been characterized and they generally consist of glycine-enriched sequences that assemble into less crystalline regions of the materials, and poly(alanine) or poly(glycine–alanine) repeats that form b-sheet crystalline structures. For recent reviews on silk structures see Heim et al. (2009), Heidebrecht and Scheibel (2013), and Eisoldt et al. (2012). Reeled silkworm silk (Bombyx mori) has been used extensively in the textile industry for over 5000 years. Sericin is extracted from the cocoons by immersion in hot soapy water, leaving 300 to 1200 m of usable fiber. Unlike silkworm silks, spider silk production has not been domesticated for textile material applications. This is because spiders are more difficult to raise in large numbers, owing to their solitary and predatory nature, and because orb webs which contain up to five different types of silk are not reelable as a single fiber like the cocoon silk from the silkworm. Further, in comparison to the silkworm, spiders generate only small quantities of silk. Despite the intriguing mechanical features of spider silks, the difficulty in domesticating spiders necessitates an alternative approach to silk production: genetic engineering and recombinant production.
2
Silk Processing
The processing of water-soluble high molecular weight silk proteins into water-insoluble fibers involves many factors, including acidification, salting out/phase separation, and perhaps other chemical or physical steps. The vast majority of silk proteins is large and consists of a core repetitive region, being made up of sequence motifs that can be repeated several hundred times (Guerette et al., 1996; Hayashi and Lewis, 1998, 2000). This core region imparts the physical properties onto the resulting silk thread. Most of the proteins then have non-repetitive terminal sequences with a unique fold in the protein. In the case of Major Ampullate spider silk proteins it has been shown that the termini act as a switch being involved in the storage of the proteins and triggering the protein to convert from a soluble to an insoluble fiber with aligned crystalline b-sheet regions (Eisoldt et al., 2012). In the spinning duct, changes in physiological conditions such as pH and salt concentrations accompany and further trigger the structural conversion/assembly, and physical shear generated during spinning finalizes silk fiber processing (Jin and Kaplan, 2003; Heim et al., 2009; Eisoldt et al., 2011). The highly organized fibrous structure of silk and the extensive hydrogen bonding and van der Waals interactions lead to the exclusion of water from the intersheet regions of the b-sheets after silk spinning. Most silk fibers are insoluble in water, dilute acids and alkali, chaotropic agents such as urea and guanidine hydrochloride, and most organic solvents. Silk can be solubilized by immersion of the fibers in very high concentration salt solutions such as lithium bromide, lithium thiocyanate, guanidinium thiocyanate or calcium chloride and other calcium salts. High concentrations of propionic acid/hydrochloric acid mixtures, hexafluoroisopropanol and formic acid can also be used to facilitate solubility. After solubilization, dialysis into other chaotropic agents, water or buffers can be carried out (Kaplan et al., 1994; Hardy et al., 2008). ☆
Change History: June 2016. T. Scheibel revised the article completely.
Reference Module in Materials Science and Materials Engineering
doi:10.1016/B978-0-12-803581-8.02274-8
1
2
Overview of spider silk cDNAs expressed in different host organisms
Type of silk Protein Major ampullate MaSp1
MaSp1 (fibroin chimera) MaSp1 and MaSp2
MaSp1- and MaSp2-collagen fusion protein ADF3 ADF3 and ADF4
Flagelliform Flag Polyhedron-flag fusion protein Tubiliform (aka cylindriform) TuSp1 TuSp1 Pyriform PySp2 a
Molecular weight (kDa)
Spider
Host organism
References
22, 25 10–28
Euprosthenops sp. Euprosthenops australis
Mammalian cells (COS-1) Bacteria (Escherichia coli)
43 83 12 14 60–140 31–66 33–39 57–61 60–140 60 50–105 35–56
Nephila clavipes Nephila clavata N. clavipes Latrodectus hesperus N. clavipes N. clavipes N. clavipes N. clavipes Araneus diadematus A. diadematus A. diadematus A. diadematus
Bacteria (E. coli) Transgenic animals (Bombyx mori) Bacteria (E. coli) Bacteria (E. coli) Mammalian cells (MAC-T and BHK)a Transgenic animals (mice) Yeast (Pichia pastoris) Yeast (P. pastoris) Mammalian cells (MAC-T and BHKa) Transgenic animals (goats) Insect cells (Spodoptera frugiperda) Insect cells (S. frugiperda)
35–56
A. diadematus
Insect cells (Trichopulsia ni)
Grip et al. (2006) Askarieh et al. (2010), Hedhammar et al. (2008), and Stark et al. (2007) Arcidiacono et al. (1998) Wen et al. (2010) Sponner et al. (2005) Hagn et al. (2010, 2011) Lazaris et al. (2002) Xu et al. (2007) Teule et al. (2003) Teule et al. (2003) Lazaris et al. (2002) Karatzas et al. (1999) Ittah et al. (2006) Huemmerich et al. (2004b) and Vendrely and Scheibel (2007) Vendrely and Scheibel (2007)
28 61
Araneus ventricosus A. ventricosus
Insect cells (S. frugiperda) Insect cells (S. frugiperda)
Lee et al. (2007) Lee et al. (2007)
12–15 33, 45
Nephila antipodiana L. hesperus
Bacteria (E. coli) Bacteria (E. coli)
Lin et al. (2009) Gnesa et al. (2012)
N/Ab
L. hesperus
Bacteria (E. coli)
Geurts et al. (2010)
MAC-T: bovine mammary ephithelial alveolar cells, BHK: baby hamster kidney cells. N/A: not applicable.
b
Recombinant Silk Production in Bacteria
Table 1
Recombinant Silk Production in Bacteria
3
3
Properties of Silks
The mechanical properties of silks include an intriguing combination of high strength and elasticity yielding an outstanding toughness (Cunniff et al., 1994; Xu and Lewis, 1990; Heidebrecht et al., 2015). Some spider silks exhibit over 200% elongation and others maintain tensile strengths approaching those of high performance fibers such as Kevlar. In terms of energy absorption prior to break, spider silks are unmatched in the world of synthetic or natural fibers. These properties are significantly higher for spider dragline silk than silkworm silks. The mechanical properties of silk fibers are a direct result of the size and orientation of the b-sheet (crystalline) domains, the connectivity of these domains to the amorphous domains, and the interfaces or transitions between amorphous and crystalline domains. The superior performance of spider silk in webs is, however, not due merely to its exceptional ultimate strength and strain, but arises also from the nonlinear response of silk threads to strain and their geometrical arrangement in a web (Cranford et al., 2012). Resistance to axial compressive deformation is another unique feature of these fibers observed with microscopic evaluations of knotted single fibers. No evidence of kink-band failure on the compressive side of knot curves was observed. Synthetic high performance fibers fail by this mode even at relatively low stress levels; this is a major limitation with synthetic fibers in many applications. In addition, spider silk shows a torsional shape memory that prevents the spider from twisting and turning during its descent on a silk thread (Emile et al., 2006). Interestingly, the silk fiber needs no extra stimulus for total recovery after being turned from its initial position. Instead, it scarcely oscillates after twisting because of its high damping coefficient. Spider silk also shows supercontraction (Perez-Rigueiro et al., 2003; Liu et al., 2005). Absorption of water leads to shrinkage and tightens the thread. This process is important to ensure the rigidity of the spider’s web during its lifetime and is thought to be caused by the organization and arrangement of individual silk proteins. Finally, spider dragline silk is thermally stable to about 2301C. Table 2
Overview of designed spider silk genes expressed in bacterial hosts
Type of silk Protein
Molecular weight (kDa)
Spider
Host organism
References
Major ampullate MaSp1
N/Aa 100–285 15–26
Latrodectus hesperus Nephila clavipes N. clavipes
Salmonella typhimurium Escherichia colib E. coli
45–60
N. clavipes
E. coli
MaSp1 (Gly-rich repeats) MaSp2
10–20 63–71
N. clavipes Argiope aurantia
E. coli E. coli
MaSp2/Flag MaSp1 and MaSp2
31–112 58, 62 20–56
N/A N. clavipes N. clavipes
E. coli E. coli E. coli
ADF3, ADF4
N/A 55, 67 15–41 65–163 34–134
Bacillus subtilis E. coli E. coli E. coli E. coli
ADF1, ADF2, ADF3, ADF4
25–56
N. clavipes N. clavipes N. clavipes N. clavipes Araneus diadematus A. diadematus
S. typhimurium
Widmaier et al. (2009) and Widmaier and Voigt (2010) Xia et al. (2010) Szela et al. (2000) and Winkler et al. (1999, 2000) Bini et al. (2006), Huang et al. (2007), and Wong Po Foo et al. (2006) Fukushima (1998) Brooks et al. (2008b) and Teule et al. (2009) Lewis et al. (1996) Teule et al. (2007) Arcidiacono et al. (2002) and Mello et al. (2004) Fahnestock (1994) Brooks et al. (2008a) Prince et al. (1995) Fahnestock and Irwin (1997) Huemmerich et al. (2004a) and Heidebrecht et al. (2015) Widmaier et al. (2009) and Widmaier and Voigt (2010)
N/A
N. clavipes
S. typhimurium
Flag
14–94
N. clavipes
E. coli
Flag (Gly-rich repeats)
25
N. clavipes
E. coli
Widmaier et al. (2009) and Widmaier and Voigt (2010) Vendrely et al. (2008) and Heim et al. (2010) Zhou et al. (2001)
90
Nephila antipodiana
E. coli
Oster et al. (2014)
76–89 18–36
N/A N/A
E. coli E. coli
Cappello et al. (1990) Yang and Asakura (2005)
Flagelliform silk Flag
Tubuliform/cylindriform silk TuSp1-GFP N/A N/A Gly-rich repeats and Alablocks a
N/A: not applicable. Metabolically engineered.
b
4
4
Recombinant Silk Production in Bacteria
Silk Genetics and Cloning
Recently, remarkable progress has been made in understanding silk genetics, structures and biophysics, as well as their recombinant production. Biotechnological strategies concerning silk protein production have to consider several issues such as the size of the underlying silk genes and their highly repetitive sequences. Nowadays, cloning and expression of native silk sequences (cDNA) have been achieved in a variety of host systems as summarized exemplarily for spider silk in Table 1. The use of bacterial hosts, such as Escherichia coli, typically used in industrial processes, for the production of full-length native silk proteins is limited due to a distinct codon usage different to that of spiders and due to the size-limited yield of recombinant proteins in E. coli. Furthermore, bacterial hosts often remove repetitive sequences by a mechanism of homologue recombination. Therefore, expression of (partial) silk cDNA is often ineffective in E. coli. Since the codon usage of eukaryotic organisms is more closely related to that of insects and spiders, eukaryotic cells reflect more suitable expression hosts. Successful expression of, for example, cDNA fragments from two spiders, Nephila clavipes and Araneus diadematus, was achieved in bovine mammary epithelial and hamster kidney cells, as well as in insect cells. The drawback of these systems is the lack of easy and cheap scalability to yield high amounts of silk proteins. Since bacteria are easy to handle and can be grown in industrial scales at low costs, a way was sought to overcome the described complications. Therefore, synthetic genes with optimized codon usage or variants based on consensus repeat sequences garnered from data from native genes, as well as variants combining consensus sequences with natural sequences, for example, encoding terminal domains have been engineered and expressed in bacteria (exemplarily shown for different spider silks in Table 2). The described protein engineering technique yields mimetics of spider silk as well as new proteins with additional features not found in natural silk proteins. For instance, it has been investigated whether the incorporation of specific amino acids influences protein solubility and allows triggering of silk assembly. To gain new functional properties, incorporation of amino acids with chemically active side chains, like lysine or cysteine, is also feasible, since this allows side specific functionalization of the silk proteins. Even the addition of oligopeptides is conceivable. In this context chimeric proteins consisting of a spider silk domain and a peptide known to precipitate silica or the cell recognition peptide RGD have been successfully engineered. Thus, chimeric silk proteins can comprise chemically active sites, enzymatic activity, and receptor binding sites, among other functionalities (Humenik and Scheibel, 2014; Borkner et al., 2014; Schacht and Scheibel, 2014). Importantly, bacteria are not the only suitable scalable hosts expressing engineered recombinant silks, and therefore a variety of hosts including yeast, plants, insects and even goats has been tested as shown exemplarily for spider silk in Table 3.
5
Future Directions
Novel and important applications for silk proteins can be envisioned, owing, for example, to the unique mechanical properties of the fibers made thereof (Heidebrecht et al., 2015). Further, plenty of studies have been focused on silk-based biomaterials, due to Table 3
Overview of designed spider silk genes expressed in different eukaryotic hosts
Type of silk Protein Major ampullate MaSp1
MaSp2
MaSp1 and MaSp2 Flagelliform Flag N/A Amphiphilic silk-like protein a
N/A: not applicable.
Molecular weight (kDa)
Spider
Host organism
References
N/Aa 94
Nephila clavipes N. clavipes
Yeast (Pichia pastoris) Yeast (P. pastoris)
64, 127
N. clavipes
Plants (Arabidopsis thaliana)
13–100
N. clavipes
Plants (Nicotiana tobaccum)
13–100
N. clavipes
Plants (Solanum tuberosum)
70 70 113
Insect cells (Bombyx mori) Transgenic animals (B. mori) Yeast (P. pastoris)
65 60 50
Nephila clavata N. clavata Nephila madagascariensis N. clavipes N. clavipes N. clavipes
Fahnestock and Bedzyk (1997) Agapov et al. (2009) and Bogush et al. (2009) Barr et al. (2004) and Yang et al. (2005) Scheller and Conrad (2005) and Scheller et al. (2001, 2004) Scheller and Conrad (2005) and Scheller et al. (2001, 2004) Zhang et al. (2008) Zhang et al. (2008) Bogush et al. (2009)
Plants (N. tobaccum) Plants (N. tobaccum) Transgenic animals (goat)
Patel et al. (2007) Menassa et al. (2004) Perez-Rigueiro et al. (2011)
37
N. clavipes
Insect cells (B. mori)
Miao et al. (2006)
28–32
N/A
Yeast (P. pastoris)
Werten et al. (2008)
Recombinant Silk Production in Bacteria
5
the realization that silk proteins are non-immunogenic. The option to genetically tailor composition and structure provides a basis for new types of biomaterials based on silks for a variety of biomedical needs, including sutures, implant coatings, drug delivery systems, or scaffolds for tissue engineering (Leal-Egana and Scheibel, 2010; Schacht and Scheibel, 2014). Biofabrication is a new technology to simultaneously 3D-print materials together with cells in which silk proteins show extraordinary properties when used as bioinks (Schacht et al., 2015; DeSimone et al., 2015; Jüngst et al., 2016). However, also technical applications are envisioned in the near future such as filter devices, photonics, optical or electrical devices (Omenetto and Kaplan, 2010; Tao et al., 2012; Borkner et al., 2014).
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Biomacromolecules 7, 3139–3145. Bogush, V.G., Sokolova, O.S., Davydova, L.I., et al., 2009. A novel model system for design of biomaterials based on recombinant analogs of spider silk proteins. J. Neuro. Pharmacol. 4, 17–27. Borkner, C., Elsner, M., Scheibel, T., 2014. Coatings and films made of silk proteins. Appl. Mater. Interface 6, 15611–15625. Brooks, A.E., Nelson, S.R., Jones, J.A., et al., 2008a. Distinct contributions of model MaSp1 and MaSp2 like peptides to the mechanical properties of synthetic major ampullate silk fibers as revealed in silico. Nanotech. Sci. Appl. 1, 9–16. Brooks, A.E., Stricker, S.M., Joshi, S.B., et al., 2008b. Properties of synthetic spider silk fibers based on Argiope aurantia MaSp2. Biomacromolecules 9, 1506–1510. Cappello, J., Crissman, J., Dorman, M., et al., 1990. Genetic engineering of structural protein polymers. Biotechnol. Prog. 6, 198–202. Cranford, S.W., Tarakanova, A., Pugno, N.M., Buehler, M.J., 2012. 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Further Reading Augsten, K., Muhlig, P., Herrmann, C., 2000. Glycoproteins and skin-core structure in Nephila clavipes spider silk observed by light and electron microscopy. Scanning 22, 12–15. Schmidt, F.R., 2004. Recombinant expression systems in the pharmaceutical industry. Appl. Microbiol. Biotechnol. 65, 363–372.