Review

Spider silks and their applications

Jonathan A. Kluge, Olena Rabotyagova, Gary G. Leisk and David L. Kaplan
Departments of Biomedical Engineering, Chemistry and Mechanical Engineering, Tufts University, Medford, MA 02155, USA

Spider silks are characterized by remarkable diversity in

their chemistry, structure and functions, ranging from
orb web construction to adhesives and cocoons. These
unique materials have prompted efforts to explore
potential applications of spider silk equivalent to those
of silkworm silks, which have undergone 5000 years of
domestication and have a variety of uses, from textiles to
biomedical materials. Recent progress in genetic engineering of spider silks and the development of new chimeric spider silks with enhanced functions and specific
characteristics have advanced spider silk technologies.
Further progress in yields of expressed spider-silk
proteins, in the control of self-assembly processes and
in the selective exploration of material applications is
anticipated in the future. The unique features of spider
silks, the progress and challenges in the cloning and
expression of these silks, environmentally triggered silk
assembly and disassembly and the formation of fibers,
films and novel chimeric composite materials from
genetically engineered spider silks will be reviewed.
Introduction
Silk has captured the imagination of humankind for millenia, largely due to the unrivaled visual and functional
properties of silk fiber and the unique structures that have
been generated by various silk-producing species in
nature. These structures include amazing orb web structures spun to capture prey, cocoons to house developing
offspring, adhesives used to anchor webs and fibrous
tethers to capture flying prey. Despite significant interest
in other silk sources, such as spider silk, silkworms have
been the traditional source exploited. One reason that
spider silk has lagged behind is that silkworms are fairly
easy to domesticate, whereas spiders cannot be housed
in high densities (Encyclopdia Britannica, retrieved
December 27th, 2007, http://www.britannica.com/eb/
article-6677). In addition, whereas one silkworm cocoon
yields 600 to 900 m of fiber, only 137 m of fiber can be
reeled from the ampullate gland of a spider and only 12 m
of silk is found in a complete spider web [1]. Finally, many
types of silk are utilized in web construction from a single
spider. Therefore, the best option for moving the application of spider silks forward is the pursuit of biotechnological means of generating source material. Recent
advances in genetic engineering have led to increased
insight into silk proteins and the structural organization
of spider-silk-encoding genes. With this knowledge, heterologous expression of spider-silk proteins in a range of host
systems has been achieved, as well as the formation of new
materials from recombinant-DNA-derived spider-silk
Corresponding author: Kaplan, D.L. (David.Kaplan@tufts.edu).

244

proteins. These advances might make spider silk a viable

choice in many new application areas, heretofore the
domain of silkworm silk.
Chemistry, structure and function
The molecular structure of silk consists of regions of
protein crystals separated by less organized protein
chains. The primary structural modules give rise to diverse
secondary structures that, in their turn, direct functions of
different silks. As the most heavily studied secondary
structure of silks, crystalline b-sheets contribute to the
high tensile strength of silk fibers. Beta sheets form
through natural physical crosslinking of amino acid
sequences, which in spider and silkworm silk consist of
multiple repeats of mainly alanine, glycine-alanine, or
glycine-alanine-serine. The non-crystalline regions of silk
are commonly made up of: (i) b-spirals similar to a b-turn
composed of GPGXX repeats (where X is mostly glutamine)
and (ii) helical structures composed of GGX [2,3]. These
semi-amorphous regions provide silk with elasticity. For
example, the flagelliform silk from Nephila clavipes is rich
in the GPGXX motif, and this sequence results in a highly
elastic fiber that functions in prey capture. In addition to
the crystalline and semi-amorphous regions, non-repetitive regions are present at the amino- and carboxyl termini
of the proteins. Although the impact of these termini on
mechanical response is not fully understood, it has been
speculated that they might play a role in the controlled
assembly of silk proteins [4,5].
Each silk-producing creature synthesizes silk proteins
that offer a rich diversity of primary sequences and secondary structures. For example, the common amino acid
modules in the silk fibers synthesized by the Araneomorphae (true spiders) can be grouped into four categories:
poly-Ala, poly-Ala-Gly, GPGXX, GGX and a spacer
sequence [6]. Most recently, Garb and colleagues characterized six novel silk proteins from the Mygalomorphae
(tarantulas) that do not contain these four categories found
in true spider spidroins. These newly-characterized tarantula silks, as a result, do not possess high tensile
strength and elasticity. This finding supports the hypothesized role of poly-Ala and GPGXX modules in forming
dragline and flageliform silks [7].
In most of the above cases, the fundamental process of
silk protein self-assembly into functional materials
remains consistent, with the more hydrophobic domains,
mainly the alanine, glycine-alanine and glycine-alanineserine repeats, driving the process. In most spider silks, bsheet formation is achieved in a spinning duct caused by
the progressive loss of water in the gland and alignment of
the hydrophobic regions during flow (Figure 1) [812].
Exceptions are the more hydrophilic silks, such as those

0167-7799/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2008.02.006 Available online 25 March 2008

Review

Trends in Biotechnology

Vol.26 No.5

Figure 1. Structural hierarchy in silk assembly related to assembly into fibers. (a) (i) Spider-silk proteins consist of repeats of amino acid sequences that self-assemble into
b-sheets. This self assembly is driven by hydrogen bonding and also by hydrophobic regions. These interactions result in the formation of inter- or intra-molecular protein
chain interactions. The b-sheet structures further assemble into soft micelles in a manner that excludes the hydrophilic ends to the perimeter. The interiors of the micelles
contain water (blue regions in the figure) due to the presence of small spacers that are more hydrophilic than the dominant hydrophobic domains. This does not represent
a multi-shell structure; rather, it is caused by the partitioning of the hydrophilic chain ends to the surface of the micelles and the location to the interior of the large and
dominant hydrophobic domains and small hydrophilic spacers. With increasing protein concentration, micelles transform into gel-like states leading to metastable liquid
crystalline structures. (ii) Triggers, such as physical shearing, or environmental factors, such as low pH, methanol, ultrasonication and electric fields, convert the gel states
and liquid crystals into a more stable b-sheet structure. The resulting fibrils emerging from spinning ducts are combined into higher ordered structures as naturally
constructed webs or cocoons. (b) Molecular structure of a spider-silk protein. In silks that are used for web architecture and other strong fibers, the underlying molecular
structure consists mainly of hydrophobic regions (illustrated by thin black lines). These large hydrophobic regions are interspersed with small hydrophilic spacers
(illustrated by gray vertical rectangles) and are flanked by nonrepetitive domains at the N- and C-termini (green horizontal rectangles).

involved in adhesion, wherein charge interactions can play

a more dominant role than the hydrophobic interactions.
The self-assembly without chemical crosslinking provides
stability while still allowing enzymatic digestion [4] or slow
degradation under appropriate environmental conditions
[13].
Sources and cloning of spider silks
The spider silks that have been most extensively studied to
date with recombinant DNA technology include those from
N. clavipes (Major Spidroin dragline types I and II MaSpI
and MaSpII), Araneus diadematus (dragline ADF-3 and
ADF-4) and N. clavipes flagelliform [1422]. Two main
approaches have been used to obtain the gene encoding
spider silks: (i) isolation of native spider silk amino acid
sequences from peptide digests, followed by back-translation into the corresponding DNA sequence and chemical
synthesis of oligonucleotides to represent the sequence and
(ii) generation of cDNAs encoding the spider silk from
mRNAs that were isolated directly from silk-producing
glands. Because spider silk sequences are formed from
repeated polypeptides, chemically synthesized oligonucleotides encoding these polypeptide units can be used
as building blocks for ligation into multiple repeats to
generate longer silk-encoding genes. The advantage of

using synthetic oligonucleotides is that codons can be

optimized for different expression systems in an attempt
to improve final protein yield. The first approach (i) was
recently successfully employed in obtaining the full length
spider silk gene sequence and flanking regions from the
black widow spider (Latrodectus hesperus) [23,24].
The elucidation of the genetic organization of spider silk
genes has led to new insights into evolutionary biology based
on their complex organization of introns and exons [6,25
29]. These insights point to the importance of these remarkable gene structures in fostering stability and high levels of
expression in the native hosts. However, this very structural
design that has served well in generating the remarkable
mechanical properties of silks remains an impediment to the
large-scale expression of spider silks in simpler host systems, such as Escherichia coli. This impediment to scaling
up has prompted efforts to explore a range of different host
systems, including yeast (e.g. Pichia pastoris), insect cells
(e.g. sf9), bovine mammary epithelial alveolar cells, baby
hamster kidney cells, transgenic plants such as Arabidopsis,
soybean, potato and tobacco, transgenic mammals such as
mouse and goat and, finally, transgenic silkworms
[8,14,15,17,19,21,22,3033]. An important result was the
generation of transgenic mice that carried spider dragline
silk genes under control of the whey acidic protein (WAP)
245

Review
promoter [30]. These mice were able to produce ADF-3 and
MaSpI silk proteins and secreted these in their milk over
generations.
However, despite the use of the various host systems
described above, the molecular sizes of the expressed
spider-silk proteins are much smaller compared to that
of native proteins due to issues of gene stability and the
repetitive nature of the genetic sequences involved. To
date, a successful expression of full length spider silk
clones has not been achieved. To fully recapitulate silk
properties, all protein domains present in the native
proteins are thought to be crucial, and the absence of some
protein domains therefore severely affects the quality of
the resulting silk, either because of improper assembly or
loss of material properties. Commercial applications of
recombinant spider silks therefore remain limited because
of the inability to produce sufficient quantities of silk
proteins at a reasonable cost and with an accurate molecular weight. Because production costs for plant expression are estimated to be a fraction of those for bacterial
fermentation [21], transgenic plants might prove a feasible
option once the purification issue has been solved.
Processing spider silks into biomaterials
One attractive application of spider silks is to emulate the
diverse material functions of this family of proteins as a
source of novel biomaterial designs. Insight into the assembly and processing of spider-silk proteins into various
material forms has been a longstanding focus and
has allowed the broadening of the field of applications
for silks in general. Specifically, medical devices and tissue

Trends in Biotechnology Vol.26 No.5

engineering applications are perhaps the most promising

areas for the utilization of spider silks. Recent progress
with reprocessed or native silkworm silk fibers has been
realized, and similar approaches could be used with spider
silks when they become available in sufficient quantities.
For example, in ligament tissue engineering, a combination of fiber twisting and braiding of silkworm silk fibers
was able to direct stem-cell- and ligament-cell-based reconstruction through the alignment and mechanical
strength of the twisted silk structure [34,35]. Recently,
spider silk fibers were manually collected from the major
ampullate dragline and seeded with human Schwann cells
to demonstrate biocompatibility, suggesting a promising
strategy for future treatment of peripheral nerve injuries
[36].
Various studies have been conducted to assess the
solubility and solution structure of genetically engineered
spider silks, including variants of ADF-3 and ADF-4 that
adopted a random coil structure similar to analogs of major
ampullate gland proteins in N. clavipes [37]. Different
secondary structure results were obtained for protein analogs of MaSpI [16]. To control solubility of spider-silk
proteins, genetically engineered variants have been generated with environmentally regulated molecular triggers
based on either chemical or biochemical reactions
(Figure 2) [3841]. These systems were based on N. clavipes dragline silk analogs, one with the insertion of methionine residues to serve as redox triggers and the other
based on the inclusion of an enzymatic phosphorylation
site for cyclic AMP-dependent protein kinase phosphorylation. Both systems were reversible (oxidation/reduction;

Figure 2. Molecular triggers for environmental control of b-sheet assembly. To control assembly of spider silks, different modes of genetic modification have been
employed that allow a controllable assembly or disassembly of silk b-sheets. (a) The consensus repeat of spider dragline silk from N. clavipes is shown. b-sheet forming
regions are indicated by the brick wall icon, which denotes their insoluble state when assembled. (b) A phosphorylation site was introduced by the addition of two new
codons into the sequence to provide a suitable recognition site for kinase reaction. This modification generated a controllable b-sheet forming region, based on the
presence of the kinase. The phosporylation prevented assembly of the silk protein, whereas dephosphorylation by a phosphatase promoted the assembly of the protein into
b-sheets. (c) Introduction of methionine encoding sequences near the b-sheet-forming region generated an oxidation/reduction trigger for the formation of a b-sheet region.
Upon chemical oxidation of the methionine side chain, b-sheet formation was prevented, while chemical reduction of the side chain promoted b-sheet formation.

246

Review
phosphorylation/dephosphorylation). The use of molecular
triggers provides options to regulate the assembly of spider
silks for both fundamental biophysical studies and for
potential biomaterial applications.
Formation of novel biomaterials from recombinant
spider-silk proteins
Silk proteins have been shown to solubilize in water,
organic solvents and ionic liquids, indicating the versatile
options available, and they can then be processed into new
biomaterials, including fibers, films, gels, porous sponges
and other related systems (Figure 3) [37,42]. The resulting
structures and functions of the obtained materials are
directly controlled by the content and distribution of crystalline b-sheets, a process that can be controlled by the
mode of processing. A variety of environmental factors,
including solvent, pH, water, concentration of protein and
salt, influence the processing and assembly of spider silk in
vivo and in vitro [40]. Attempts to process spider-silk
proteins into distinct classes of functional materials have
been underway since the first successful expression systems were realized. Highlighted below are advances in the
field that were based primarily on the processing of genetically engineered proteins derived from spider silk, with
insight from silkworm silks serving as a starting point.
Fibers and textiles
The combination of high strength, toughness and light
weight makes spider silks attractive for high-performance
fiber and composite applications [43] and for biomedical
applications. Whether the silk material is to be used as an
individual fiber or woven into a textile structure, it
is critical that production techniques are developed to

Trends in Biotechnology

Vol.26 No.5

generate long lengths of material in sufficient quantity

and with mechanical performance that is at least equal to
the native spider silks. Traditionally, silkworm silks have
been the focus of research to generate silk fiber materials.
Techniques to form fibers from silkworm proteins, such as
solvent extrusion, electrospinning and microfluidic
approaches, might be appropriate for spider-silk proteins
as well (Figure 4). The advantages and limitations of each
system might determine their use in specific applications
or their commercial exploitation.
Silkworm silk solutions can be solubilized in aqueous
solvents, including LiBr [44] and N-methylenemorpholineN-oxide (NMMO) [45], and subsequently formed into fibers
by a wet solvent-based process [46,47]. However, relatively poor fiber mechanical properties and brittle behavior
were found when compared to native fibers (400 MPa
compared to upwards of 700 MPa reported for native B.
mori silks). Recent efforts using spider silk, however, have
shown promise. Spider silk fibers have been formed
through the electrospinning of spin dopes prepared by
dialyzing genetically engineered silk (MaSpI protein analogs) in urea containing Tris buffer and salts. This aqueous
method has generated fibers that are 1060 mm in
diameter and that exhibit molecular orientation and mechanical properties similar to natural spider silk [43]. Electrospinning of spin dopes derived from ADF-3 recombinant
spider-silk protein has generated fiber diameters up to
40 mm. Although fiber toughness and modulus compared
favorably to native A. diadematus dragline silk, tenacity
was lower [14]. Electrospinning was also used to generate
fibers with an average diameter of 300 nm from N. clavipes
MaSpI silk dope prepared in HFIP (hexafluoroisopropanol). It was shown that control of conformation can be

Figure 3. Processing of silk into new biomaterials. Silk fibers, such as native spider silks, silkworm silks and recombinant silk proteins, can be solubilized with a variety of
solvents, including formic acid, HFIP, calcium nitrate, lithium salts or ionic liquids. Once solubilized, the silk protein solutions can be processed into the range of different
structures shown. Portions of the figure were adapted from [35] and are reprinted with permission of Elsevier Limited.

247

Review

Trends in Biotechnology Vol.26 No.5

Figure 4. Schematic representation of current silk fiber-forming techniques. (a) Solvent extrusion is performed by drawing the fiber through a coagulation bath in a
controlled manner. Microfluidic devices use a contracting channel and multiple solvent inputs to regulate the geometry and chemistry of the resulting fiber. Electrospinning
processes combine strong voltage gradients and syringe pump extrusion and result in either random or aligned fiber deposition. (b) Characteristics of the different fiberforming processes. To date, electrospinning has proved to be the most useful approach, mainly because of the low amounts of spider silk materials needed and the utility of
the resulting electrospun fiber mats for cell- and tissue-culture studies. Portions of the figure were adapted from [46] and are reprinted with permission of John Wiley &
Sons, Inc.

achieved and that molecular alignment might be possible,

potentially offering improved fiber properties [48].
Films
Films have been cast using HFIP solutions that contain
recombinant forms of the dragline spider-silk proteins
ADF-3 and ADF-4. The transparent films, ranging in
thickness from 0.5 to 1.5 mm, could be made water-insoluble through the use of potassium phosphate or methanol,
which converted the proteins secondary structure from ahelix to b-sheet. When this treatment was applied to the
recombinant form of the ADF-4 protein, a level of chemical
stability in certain denaturants was achieved that rivaled
the stability of native dragline silk. Cloning strategies and
the ability to modify the film surfaces for attachment of
functional molecules make these materials promising for
applications such as wound dressings and enzyme immobilization scaffolds [49,50].
Hydrogels and porous sponges
Hydrogels are utilized in the field of tissue engineering to
form porous but stable tissue scaffolds. By adding methanol to recombinant forms of the dragline silk protein ADF4, the silk has been shown to self-assemble into nanofibers
with diameters of 3 nm and lengths less than 1 mm. Over
the course of a few days, these nanofibers transformed into
a hydrogel fiber network. These hydrogels exhibited a non248

linear viscoelastic material response with low stiffness and

strength. Cross-linking was induced by visible light after
applying ammonium peroxodisulfate and tris(2,20 -bipyridyl)dichlororuthenium (II) to the surface of the hydrogel.
Cross-linked hydrogels demonstrated fairly linear
material response with much greater modulus and
strength response. Given the superior mechanical
response and the stability of the hydrogel over weeks,
these materials are suitable for tissue engineering applications [51]. Sponge-like, porous three-dimensional structures are also important in tissue engineering. Such
structures act as a scaffold to support cells, allow transport
of nutrients and metabolic wastes and promote tissue
development [52,53]. While development of silkwormsilk-based sponges has progressed for such applications,
the use of spider silk has lagged behind. In one application
of spider silk, spider cocoon silk was used to create porous
scaffolds for tissue engineered cartilage. Through a process
of solubilizing of the cocoon silk in lithium bromide (LiBr),
mixing with salts and methanol treatment, porous scaffolds were generated that sustained chondrocyte cell
growth, which could lead to articular cartilage development [54].
Microcapsules
Microcapsules of the recombinant form of ADF-4 spider
silks were developed by controlling silk self-assembly at an

Review
oilliquid interface. The resulting b-sheet-rich thin
polymer shells had high mechanical stability, with wall
thicknesses on the order of 50 nm and diameters between 1
and 30 mm. In addition, the properties of the microcapsules, including constrained degradation response to tissue-specific enzymes, were easily controlled. These
materials, therefore, offer promise for a range of applications, from drug delivery to microreactor design [55].
Functionalization of spider silk novel composites
Surface functionalization is an important strategy for
modifying the exterior of biomaterials to influence cell
and tissue development, and this strategy has been applied
to silk surface chemistry [42]. Surface modification usually
targets carboxylic acid groups on the amino acids in the
protein. Silkworm fibroin silk has been modified by
coupling enzymes such as horseradish peroxidase, cell
binding domains such as Arg-Gly-Asp (RGD) peptides
and cell signaling factors such as parathyroid hormone
(PTH) and bone morphogenetic protein-2 (BMP-2)
[44,56,57] to improve cell interactions and function or to
successfully form gradients on the silk material surface.
However, in silkworm silk fibroin only 3.3% of the amino
acids have carboxyl side groups, whereas this number is
significantly higher in spider silks, which offer the
advantage of forming higher degrees of substitution with

Trends in Biotechnology

Vol.26 No.5

cell-modifying functional groups. For example, MaSp1 has

11.7% of aspartic acid and glutamic acid residues, which
are theoretically available for chemical modifications [58].
So far, the dragline silks ADF-3 and ADF-4 from the
garden spider Araneus diadematus have been functionalized by coupling films formed from the proteins with
fluorescein and b-galactosidase using carbodiimide chemistry via EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide) [49]. This approach offers high levels of surface
decoration with enzymes, which could be used for biosensors and reactive media and to influence cell and tissue
functions. Genetically engineered spider silks can offer the
added benefits of providing designed functionalities
encoded during gene construction. Strategies employed
for spider silks have included the incorporation of cell
binding domains, such as RGD [59], or domains that
interact selectively with inorganic components to generate
novel organicinorganic composite material systems
[60,61], where N. clavipes provides the spider silk component. Highly tailored hybrid or chimeric spider silks
containing cell integrin binding domains (RGD) [59] and
domains that were able to selectively undergo mineralization with silica [60] and hydroxyapatite [61] were generated (Figure 5). The composite morphology and structure
could be manipulated by controlling processing conditions
to produce films and fibers. The control over the size of the

Figure 5. Generation of chimeric silk proteins as a tool to expand their functional features. (a) The consensus spider silk sequence that constitutes the core repeats of
chimera is shown. (b) Examples of protein regions used for the generation of chimeric silk proteins. The R5 component of silaffin (sequence shown) was fused to spider
dragline silk and was able to foster the polymerization of silica precursors to form glassified materials. The C-terminal domains from dentin matrix protein 1 (DMP 1), a
protein found in the mineralized tissue of teeth and responsible for the nucleation and growth of hydroxyapatite, could also be fused to the spider silk and was shown to
nucleate and form hydroxyapatite-containing spider silk in film form. The third example shows the addition of a cell binding domain, RGD from fibronectin, which is
responsible for cell binding via membrane integrin receptors. This resulted in cell adhesion on silk films. The last example shows the formation of block copolymers formed
from silkworm silk and elastin for biomaterials used as drug delivery systems. (c) The basic cloning procedure involving synthetic oligonucleotides is illustrated; this
procedure leads to the purifcation of silk proteins derived from recombinant DNA that have new functions. These novel proteins can be used to generate silk materials with
improved functionalities, such as improved cell binding features, glassified silk fibers with increased stiffness and hydroxyapatite mineralized biomaterials
potentially useful for bone-related repairs. Portions of the figure were adapted from [60] and [61] and are reprinted with permission of Elsevier Limited and Proc. Natl.
Acad. Sci. U. S. A., respectively. The spider picture was taken by Josh Hillman and Emily Earp (http://FLNature.org/).

249

Review
inorganic domains, as well as the molecular-level
interactions between the organic and inorganic domains
in these composites, offers a biomimetic approach toward
tougher and stiffer silk composite materials that might be
potentially useful in hard tissue remodelling or in adhesive
fillers. These results suggest that chimeric spider-silk
proteins might provide new uses for spider silk in biomedical and other specialty materials. Further benefits of this
approach might arise due to the ability to process and
assemble these novel composite materials in aqueous
ambient processing conditions.
Conclusions
The path ahead for spider-silk proteins remains challenging, mainly due to the inadequate supply of full length
proteins that are required for a full exploration of the most
interesting applications. Thus, currently, the more readily
available silkworm silk is most widely applied in the
biomedical area. Over the last decade there has been
considerable progress in understanding the gene organization of spider silk. The cloning and expression techniques
for spider silks have been improved, and the self-assembly
and processing of spider silk into many material formats is
now better understood. However, further achievements are
required, such as the expression of full length spider silk
genes to fully embrace the complex sequence structure
important for accurate assembly and function of spider
silks, as well as the establishment of efficient protein
purification protocols, especially from transgenic plants.
It will also be important to further understand and exploit
the processing of these proteins in aqueous environments
to fully recapitulate the native process used by spiders to
organize these proteins into functional materials. This is
essential because b-sheet formation dictates the resulting
material properties, and their features are influenced
directly by the presence of water and the rate of water
removal during formation of the protein into biomaterials.
An important benefit of using spider silks is their range
of material characteristics, which arise from the extensive
array of spider-silk proteins that are used throughout
the normal life-cycle of these organisms. This is a feature
that distinguishes spider silk from B. mori silkworm silk,
which contains only one single type of silk.
The influence of genetic variation, and thus protein
chemistry, on the material properties and applications
for different spider silks needs to be further investigated.
One path to achieving this improved understanding is via
the use of synthetic genes to encode the key sequences in
the various silks for their assembly into functional
materials. Full length clones or synthetic analogs that
encompass all key sequence components in a spider silk
will be required for these studies. Furthermore, these full
length clones will facilitate improved aqueous processing
and the assembly into different materials originating from
these proteins. Achieving this goal will open the door to
the use of these diverse spider-silk protein systems in a
range of novel applications, from glues to tougher composite materials and lightweight webbing. At present,
novel hybrid or chimeric spider silk systems have been
developed that are feasible for a variety of biomedical
processes, such as the use of silk-inorganic composites to
250

Trends in Biotechnology Vol.26 No.5

regulate cellmaterial interfacial interactions, to control

bonding to tissues and to modulate degradation profiles.
These applications are expected to be realized in the near
future because only relatively low quantities of silk
proteins are needed. However, applications of spider silks
in commodity materials, such as textiles, high performance
composite materials and durable and tough materials in
general, will require the success of robust and low cost
expression systems.
The development of a range of recombinant spider silks,
possibly generated in a combinatorial mode, would offer
unique insights into a variety of processes, such as protein
preparation and protein polymer processing, with potential for improved biomaterial designs. Furthermore, it
remains to be seen whether silkworm silk variants could
also be produced to fully exploit the range of designs that
have evolved in nature, such as orb webs, cocoons and
glues. The ability to mimic these features through genetic
engineering and protein processing offers possibilities for
expanding the variety of biologically derived materials
that can be applied for medical and non-medical uses.
Interest in silk has increased with a near explosion of
biomaterials research and applications, and other natural
silk sources, such as honeybees, hornets and solitary
wasps, have also attracted attention. For example, it has
been shown that honeybee silk is composed of coiled-coil
proteins. This results in characteristics that are different
from b-sheet silks, such as differences in processing and
assembly options and in the resulting material properties
(e.g. increased toughness) [62].
Further exploitation of the aqueous processing and
assembly of highly hydrophobic silk proteins into material
structures can also be considered as a path into green
chemistry in a broader context. Because the silk proteins,
as highly hydrophobic polymers, can be processed,
assembled and formed into robust materials using only
water, this sets out a path for the exploration of other
hydrophobic polymers in a similar fashion, even if they are
of purely synthetic origin. Thus, spider silk has the potential to have an impact not only on materials science and
engineering but also on green chemistry and biomedicine
after anticipated scientific and engineering progress is
achieved.
Acknowledgements
We thank our many collaborators, students and colleagues who have been
instrumental in the studies described in part here. Grant support on silkrelated systems is greatly appreciated and has included, at various times,
the National Institutes of Health, the National Science Foundation and
the Air Force Office of Scientific Research. Special thanks go to Andy
Lovinger and Hugh Delong for their foresight and support.

References
1 Lewis, R. (1996) Unraveling the weave of spider silk. Bioscience 46,
636638
2 Kaplan, D. et al., eds (1994) Silk Polymers: Materials Science and
Biotechnology, American Chemical Society
3 Kaplan, D.L. et al. (1997) Silk. In Protein-Based Materials (McGrath, K.
and Kaplan, D.L., eds), pp. 103131, Birkhauser
4 Scheibel, T. (2004) Spider silks: recombinant synthesis, assembly,
spinning, and engineering of synthetic proteins. Microb. Cell Fact. 3, 14
5 Van Beek, J.D. et al. (2002) The molecular structure of spider dragline
silk: folding and orientation of the protein backbone. Proc. Natl. Acad.
Sci. U. S. A. 99, 1026610271

Review
6 Hayashi, C.Y. et al. (1999) Hypotheses that correlate the sequence,
structure, and mechanical properties of spider silk proteins. Int. J. Biol.
Macromol. 24, 271275
7 Garb, J.E. et al. (2007) Expansion and intragenic homogenization of
spider silk genes since the triassic: evidence from mygalomorphae
(tarantulas and their kin) spidroins. Mol. Biol. Evol. 24, 24542464
8 Huemmerich, D. et al. (2004) Novel assembly properties of recombinant
spider dragline silk proteins. Curr. Biol. 14, 20702074
9 Vollrath, F. and Knight, D.P. (2001) Liquid crystalline spinning of
spider silk. Nature 410, 541548
10 Jin, H.J. and Kaplan, D.L. (2003) Mechanism of silk processing in
insects and spiders. Nature 424, 10571061
11 Bini, E. et al. (2004) Mapping domain structures in silks from insects
and spiders related to protein assembly. J. Mol. Biol. 335, 2740
12 Exler, J.H. et al. (2007) The amphiphilic properties of spider silks are
important for spinning. Angew. Chem. Int. Ed. Engl. 46, 35593562
13 Horan, R.L. et al. (2005) In vitro degradation of silk fibroin.
Biomaterials 26, 33853393
14 Lazaris, A. et al. (2002) Spider silk fibers spun from soluble
recombinant silk produced in mammalian cells. Science 295, 472476
15 Fahnestock, S.R. and Irwin, S.L. (1997) Synthetic spider dragline silk
proteins and their production in Escherichia coli. Appl. Microbiol.
Biotechnol. 47, 2332
16 Prince, J.T. et al. (1995) Construction, cloning, and expression of
synthetic genes encoding spider dragline silk. Biochemistry 34,
1087910885
17 Fahnestock, S.R. and Bedzyk, L.A. (1997) Production of synthetic
spider dragline silk protein in Pichia pastoris. Appl. Microbiol.
Biotechnol. 47, 3339
18 Arcidiacono, S. et al. (1998) Purification and characterization of
recombinant spider silk expressed in Escherichia coli. Appl.
Microbiol. Biotechnol. 49, 3138
19 Yang, J. et al. (2005) High yield recombinant silk-like protein
production in transgenic plants through protein targeting.
Transgenic Res. 14, 313324
20 Zhou, C.Z. et al. (2001) Silk fibroin: structural implications of a
remarkable amino acid sequence. Proteins Struct. Funct. Genet. 44,
119122
21 Scheller, J. et al. (2001) Production of spider silk proteins in tobacco
and potato. Nat. Biotechnol. 19, 573577
22 Menassa, R. et al. (2004) Spider dragline silk proteins in transgenic
tobacco leaves: accumulation and field production. Plant Biotechnol. J.
2, 431438
23 Ayoub, N.A. et al. (2007) Blueprint for a high-performance biomaterial:
full-length spider dragline silk genes. PLoS ONE 2, e514
24 Ayoub, N.A. and Hayashi, C.Y. (2008) Multiple recombining loci encode
MaSp1, the primary constituent of dragline silk, in widow spiders
(Latrodectus: Theridiidae). Mol. Biol. Evol. 25, 277286
25 Hinman, M.B. et al. (2000) Synthetic spider silk: a modular fiber.
Trends Biotechnol. 18, 374379
26 Xu, M. and Lewis, R.V. (1990) Structure of a protein superfiber: spider
dragline silk. Proc. Natl. Acad. Sci. U. S. A. 87, 71207124
27 Guerette, P.A. et al. (1996) Silk properties determined by gland-specific
expression of a spider fibroin gene family. Science 272, 112115
28 Gatesy, J. et al. (2001) Extreme diversity, conservation, and
convergence of spider silk fibroin sequences. Science 291, 26032605
29 Lawrence, B.A. et al. (2004) Molecular and mechanical properties of
major ampullate silk of the black widow spider, Latrodectus Hesperus.
Biomacromolecules 5, 689695
30 Karatzas, C.N. et al. (2003) High-toughness spider silk fibers spun from
soluble recombinant silk produced in mammalian cells. In
Biopolymers. Polyamides and Complex Proteinaceous Materials II
(Fahnestock, S.R. and Steinbuchel, A., eds), pp. 500510, WileyVCH
31 Barr, L.A. et al. (2004) Production and purification of recombinant
DP1B silk-like protein in plants. Mol. Breed. 13, 345356
32 Lee, K.S. et al. (2007) Molecular cloning and expression of the Cterminus of spider flagelliform silk protein from Araneus
ventricosus. J. Biosci. 32, 705712
33 Miao, Y. et al. (2006) Expression of spider flagelliform silk protein in
Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus
expression system. Appl. Microbiol. Biotechnol. 71, 192199

Trends in Biotechnology

Vol.26 No.5

34 Altman, G.H. et al. (2003) Silk-based biomaterials. Biomaterials 24,

401416
35 Altman, G.H. et al. (2002) Silk matrix for tissue engineered anterior
cruciate ligaments. Biomaterials 23, 41314141
36 Allmeling, C. et al. (2006) Use of spider silk fibres as an innovative
material in a biocompatible artificial nerve conduit. J. Cell. Mol. Med.
10, 770777
37 Huemmerich, D. et al. (2004) Primary structure elements of spider
dragline silks and their contribution to protein solubility. Biochemistry
43, 1360413612
38 Valluzzi, R. et al. (1999) Methionine redox controlled crystallization of
biosynthetic silk spidroin. J. Phys. Chem. B 103, 1138211392
39 Szela, S. et al. (2000) Reduction-oxidation control of beta-sheet
assembly in genetically engineered silk. Biomacromolecules 1, 534542
40 Foo, C.W.P. et al. (2006) Solution behavior of synthetic silk peptides
and modified recombinant silk proteins. Appl. Phys. A 82, 193203
41 Winkler, S. et al. (2000) Controlling b-sheet assembly in genetically
engineered silk by enzymatic phosphorylation/dephosphorylation.
Biochemistry 39, 1273912746
42 Vepari, C. and Kaplan, D.L. (2007) Silk as a biomaterial. Prog. Polym.
Sci. 32, 9911007
43 Arcidiacono, S. et al. (2002) Aqueous processing and fiber spinning of
recombinant spider silks. Macromolecules 35, 12621266
44 Sofia, S. et al. (2001) Functionalized silk-based biomaterials for bone
formation. J. Biomed. Mater. Res. 54, 139148
45 Xu, Y. et al. (2005) Studies on spinning and rheological behaviors of
regenerated silk fibroin/N-methylmorpholine-N-oxide H2O solutions.
J. Mater. Sci. 40, 53555358
46 Corsini, P. et al. (2007) Influence of the draw ratio on the tensile and
fracture behavior of NMMO regenerated silk fibers. J. Polym. Sci. [B]
45, 25682579
47 Zuo, B. et al. (2007) Effect on properties of regenerated silk fibroin fiber
coagulated with aqueous methanol/ethanol. J. Appl. Polym. Sci. 106,
5359
48 Stephens, J.S. et al. (2005) Effects of electrospinning and solution
casting protocols on the secondary structure of a genetically
engineered dragline spider silk analogue investigated via fourier
transform Raman spectroscopy. Biomacromolecules 6, 14051413
49 Huemmerich, D. et al. (2006) Processing and modification of films made
from recombinant spider silk proteins. Appl. Phys. A 82, 219222
50 Junghans, F. et al. (2006) Preparation and mechanical properties of
layers made of recombinant spider silk proteins and silk from silk
worm. Appl. Phys. A 82, 253260
51 Rammensee, S. et al. (2006) Rheological characterization of hydrogels
formed by recombinantly produced spider silk. Appl. Phys. A 82, 261
264
52 Kim, U.J. et al. (2005) Three-dimensional aqueous-derived biomaterial
scaffolds from silk fibroin. Biomaterials 26, 27752785
53 Nazarov, R. et al. (2004) Porous 3-D scaffolds from regenerated silk
fibroin. Biomacromolecules 5, 718726
54 Gellynck, K. et al. (2005) Chondrocyte growth in porous spider silk 3Dscaffolds. Eur. Cell. Mater. 10 (Suppl. 2), 45
55 Hermanson, K.D. et al. (2007) Engineered microcapsules fabricated
from reconstituted spider silk. Adv. Mater. 19, 18101815
56 Karageorgiou, V. et al. (2004) Bone morphogenetic protein-2 decorated
silk fibroin films induce osteogenic differentiation of human bone
marrow stromal cells. J. Biomed. Mater. Res. A 71, 528537
57 Vepari, C.P. and Kaplan, D.L. (2006) Covalently immobilized enzyme
gradients within three-dimensional porous scaffolds. Biotechnol.
Bioeng. 93, 11301137
58 McGrath, K. and Kaplan, D., eds (1997) Protein-Based Materials,
Birkhauser
59 Bini, E. et al. (2006) RGD-functionalized bioengineered spider dragline
silk biomaterial. Biomacromolecules 7, 31393145
60 Wong Po Foo, C. et al. (2006) Novel nanocomposites from spider silksilica fusion (chimeric) proteins. Proc. Natl. Acad. Sci. U. S. A. 103,
94289433
61 Huang, J. et al. (2007) The effect of genetically engineered spider silkdentin matrix protein 1 chimeric protein on hydroxyapatite nucleation.
Biomaterials 28, 23582367
62 Sutherland, T.D. et al. (2007) Conservation of essential design features
in coiled coil silks. Mol. Biol. Evol. 24, 24242432

251