ABSTRACT

Spider silk is a unique material known for its diverse chemistry, structure, and functions.
These silks are essential for a variety of biological purposes, including web construction,
prey capture, and the formation of protective cocoons. Unlike silkworm silk, which has
been domesticated for over 5,000 years for textiles and biomedical applications, spider
silk offers an even greater potential for a wider range of uses due to its remarkable
mechanical properties. The application of spider silk in various fields, from medical
devices to advanced materials, has generated significant interest. Advances in genetic
engineering have enabled the production of chimeric spider silks, which combine the
strengths of various types of spider silk proteins to enhance their properties. Key areas
of research focus on increasing the yield of spider silk proteins, optimizing the self-
assembly processes, and improving material properties for specific applications.
Additionally, novel approaches in cloning and expressing spider silk genes have opened
up possibilities for sustainable and scalable production of these materials. This review
explores the ongoing challenges and progress in the development of spider silks,
particularly in areas such as silk assembly, fiber formation, and the creation of new
composite materials, offering a glimpse into the future of spider silk technologies (Garb
et al., 2009; Rushing et al., 2022).

Introduction

Silk has captured the imagination of humankind for millennia, 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 (Encyclopedia Britannica, retrieved December 27th,

2007, http://www.britannica.com/eb/article-6677). In addition, whereas one silkworm

cocoon yields 600 to 900m of fiber, only ∼137m of fiber can be reeled from the
ampullate gland of a spider and only ∼12m of silk is found in a complete spider

web 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 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 β-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. 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.

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 . 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.

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-alanine-serine repeats, driving the process. In most

spider silks, β-sheet 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.

Exceptions are the more hydrophilic silks, such as those 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 or slow degradation under appropriate environmental conditions

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. Two main approaches have been used to obtain the gene

encoding spider silks. 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 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 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)

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. 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.

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 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. 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.

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 different secondary structure results were obtained for protein analogs of

MaSpI. 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

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. The resulting structures and functions of the obtained materials are directly

controlled by the content and distribution of crystalline β-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. 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 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 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. The advantages and limitations of each

system might determine their use in specific applications or their commercial

exploitation.

Spider Silk Considered New Materials

Spider silk is considered a new material because of its unique properties and the

emerging potential to use it in various advanced applications, many of which were

previously limited to traditional materials like steel, plastic, or even natural fibers such as

silkworm silk. Here are several key reasons why spider silk is viewed as a "new

material" in the context of materials science and biotechnology:

1. Unmatched Mechanical Properties

Spider silk has an extraordinary combination of strength, toughness, and

elasticity, which makes it a remarkable material. It is stronger than steel by weight and
tougher than Kevlar, the material used in bulletproof vests. These exceptional properties

are due to its unique molecular structure:

 High tensile strength: Spider silk can stretch and absorb force without breaking.

 Lightweight: It is extremely light, which is important for applications where

weight is critical, such as in aerospace and medical devices.

 Elasticity: The silk can stretch up to five times its original length without

breaking, allowing it to absorb large amounts of energy.

2. Biocompatibility

Spider silk is biocompatible and non-toxic, making it ideal for use in biomedical

applications. This includes potential uses in:

 Sutures and surgical threads: Spider silk could replace synthetic materials in

stitches, offering better healing properties due to its natural origin.

 Tissue engineering: Its ability to mimic natural extracellular matrices makes it a

candidate for scaffolds in tissue regeneration.

 Drug delivery: Its fine structure could be used for controlled release of drugs in

the body.

3. Versatility in Applications

Spider silk can be processed into various material forms:

 Fibers: For textiles, composites, or lightweight structural materials.

  Films: Thin, flexible layers that can be used for biomedical coatings or as

packaging material.

 Gels and sponges: For wound dressings or tissue regeneration scaffolds.

Its versatility is also enhanced by the ability to engineer spider silk proteins to

exhibit different physical properties, such as varying stiffness, strength, or

hydrophobicity. This makes it adaptable for diverse applications, from medical devices

to eco-friendly alternatives for plastic materials.

4. Sustainability and Eco-Friendliness

As a natural polymer, spider silk is considered more environmentally friendly

compared to synthetic alternatives, especially in the context of global sustainability

concerns. The production of spider silk via biotechnology (e.g., genetically engineered

bacteria or plants) can reduce reliance on petrochemical-based materials and fossil

fuel-dependent industries. Additionally, it is biodegradable, so products made from

spider silk won't contribute to long-term pollution.

5. Biotechnological Advancements

The production of spider silk is challenging because spiders do not produce silk

in large enough quantities for industrial use. However, with recent advancements in

genetic engineering, scientists have been able to produce spider silk proteins in

bacteria, yeast, and even plants. This has opened the door to large-scale

production, enabling spider silk to be considered a viable material for industrial

applications.
 The development of chimeric silks (combining spider silk proteins with other

materials) has further expanded its potential uses, allowing for the creation of materials

with custom properties that would not be possible with natural spider silk alone.

6. Novel Functionalities

Spider silk is not just a passive material—it has functional properties that make

it valuable for specific technological innovations. For instance:

 Self-healing: Spider silk can potentially heal itself if it gets damaged, mimicking

the behavior of natural biological systems.

 Bio-inspired design: The structure of spider silk can be mimicked in synthetic

materials to create new composites with properties like self-assembly and

adaptive behavior in response to external conditions (e.g., changes in

temperature or humidity).

7. Research and Development Potential

Spider silk research is still in its early stages compared to more conventional

materials. As biotechnology advances, there is growing potential for genetically

engineered spider silks to fulfill a wide range of roles that were previously reserved for

synthetic polymers or animal-based materials. This includes:

 Space exploration: Spider silk's combination of high strength and light weight

makes it a candidate for aerospace materials.

 Wearable technology: The flexibility and strength of spider silk may allow it to

be used in smart textiles, such as clothing embedded with sensors.

Conclusions

The development of spider-silk proteins faces challenges, primarily due to the

limited availability of full-length proteins necessary for exploring their most promising

applications. As a result, silkworm silk, which is more accessible, is currently more

widely used in biomedical applications. In recent years, advances have been made in

understanding spider silk gene organization, cloning, expression techniques, and the

self-assembly of these proteins into various material formats. However, further progress

is needed, such as expressing full-length spider silk genes and improving protein

purification, particularly from transgenic plants.

Understanding the processing of these proteins in aqueous environments, which

is essential for forming β-sheets that determine material properties, is also crucial.

Spider silk's diversity of material characteristics, arising from the variety of proteins used

throughout a spider's life, distinguishes it from the single-type silk produced by

silkworms. Exploring genetic variation and protein chemistry in different spider silks,

potentially through synthetic genes, could lead to novel applications ranging from glues

to stronger composites and lightweight webbing.

Although spider silk's application in biomedical processes, such as silk-inorganic

composites, is already feasible, using it for large-scale materials like textiles and

durable composites will require robust, cost-effective expression systems. The

development of recombinant spider silks could improve biomaterial designs, and

silkworm silk variants might also be engineered to mimic diverse natural designs, such
as orb webs and cocoons. Other natural silks, such as those from honeybees, have

distinct properties and are also of interest for material science. Spider silk's aqueous

processing has potential implications for green chemistry, as its formation into materials

using water could inspire similar techniques for synthetic hydrophobic polymers.

Overall, spider silk has the potential to impact materials science, green chemistry, and

biomedicine, pending further scientific and engineering advancements.

References

Lewis, R. (1996). Unraveling the weave of spider silk. Bioscience, 46(10), 636–638.
https://doi.org/10.2307/1312947

Kaplan, D. L. (Ed.). (1994). Silk polymers: Materials science and biotechnology.

American Chemical Society.

McGrath, K., & Kaplan, D. L. (n.d.). Silk. In Silk polymers: Materials science and
biotechnology. American Chemical Society.

Scheibel, T. (1997). Protein-based materials. In Protein-based materials (pp. 103–131).

Birkhäuser.

Scheibel, T. (2004). Spider silks: Recombinant synthesis, assembly, spinning, and

engineering of synthetic proteins. Microbial Cell Factories, 3(1), 14.
https://doi.org/10.1186/1475-2859-3-14