Several publications and patent documents are referenced in this application in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and documents is incorporated by reference herein.
Plants may be viewed as model systems for the large-scale production of exogenous proteins intended for therapeutic and industrial applications. Many plants are relatively easy and inexpensive to grow and are routinely produced in large quantities. Such large quantities, or crops, are harvested and processed by standard procedures utilized by the agronomic industry. The effective use of plants as bioreactors or protein factories depends on the ability to achieve high levels of expression of exogenous protein, which is stable throughout the life cycle of the transgenic plant and whose expression is maintained in subsequent generations. Silencing of an introduced transgene may occur in transformed plants and, thus, contributes to the commercial risk involved and hampers the general economic exploitation of plants as protein factories. A number of efficient strategies have been developed to avoid transgene silencing, including careful design of the transgene construct and thorough analysis of transformants at the molecular level. Recent research has focused on additional aspects related to the generation of transgenic plants intended for protein production and their influence on the stability of heterologous gene expression (De Wilde et al. 2000. Plant Mol Biol 43:347-59).
Of note, clinical trials are proceeding on the first biopharmaceuticals derived from transgenic plants. One transgenic plant-derived biopharmaceutical, hirudin, has been commercially produced in Canada. Product purification may, however, present a potential obstacle in the process because it is expensive. Various methods have been developed to overcome this problem, including oleosin-fusion technology, which allows extraction with oil bodies. In some cases, delivery of a biopharmaceutical product by direct ingestion of the modified plant may potentially remove the need for product purification. Such biopharmaceuticals may be stored and distributed as, for example, seeds, tubers, fruits, or ground plant material. The stability of exogenous proteins expressed in plants provides an additional benefit of such systems. (Giddings et al. 2000. Nat Biotechnol 18:1151-5).
The presence of silk protein producing abdominal glands is a unique feature of spiders. Spiders are also unique in the use of these silks throughout their life span and their nearly total dependence on silk for evolutionary success (14, 19). There were periods of fairly intense study of spider silk prior to World War II and in the late 1950s. Progress was relatively meager, however, particularly when compared to that related to silkworm silk. Beginning in the 1970s, interest in spider silk was revived with several papers describing physical, mechanical and chemical properties of spider silks. The composition of spider silks has been known to be predominantly protein since the 1907 studies of Fischer (5). In fact, except for the sticky spiral thread, no significant amount of any other compound but protein has been detected.
Typical spider webs are constructed from several different silks, each of which is produced in a different gland. Due to their large size and ease of study, major ampullate glands have received the most attention. Thus, most of what is known about the synthesis of silk proteins is based on studies of major ampullate glands. Morphological and histochemical studies of the other glands, however, have confirmed the conclusions drawn from research performed using major ampullate glands. Synthesis of the silk protein(s) takes place in specialized columnar epithelial cells (2). There appear to be at least two different types of cells producing protein (14), which correlates with findings that revealed the presence of two proteins in the silk from these glands. Newly synthesized protein droplets within the cell are secreted into the lumen of the gland, which serves as a reservoir of soluble silk protein.
The protein in the lumen of the gland is believed to be in a liquid crystal state (21), a structure which prevents fiber formation prior to passage through a narrow duct leading to a spinnernet. Maintenance of the liquid crystal state is likely due to physical properties related to protein structure and concentration, which serve to prevent aggregation into large protein arrays. It has been shown that silk in the lumen is not birefringent whereas silk becomes increasingly birefringent as it passes down through the duct (22). Thus, the ordered array of protein observed in the final fiber occurs during its passage through the duct. This appears to be due to the mechanical and frictional forces aligning the protein molecules and altering the secondary structure to the final fiber form. Iizuka (13) has proposed a similar mechanism for silkworm silk formation. The ability to draw silk fibers directly from the lumen of the major, minor and cylindrical glands (Hinman, M. personal comm.) implies that the physical force of drawing the solution is sufficient for fiber formation and provides experimental evidence for this mechanism. Once the fiber has reached the spinneret, a muscular valve at the exit of the spinneret is utilized to control the flow rate of the fiber and, to a small degree the fiber diameter. The silk exits the spider through the spinnerets, of which there are three pairs, anterior, median and posterior.
One of the features attracting researchers to study spider silks is their unusual mechanical properties. Orb-web weaving spiders use the minimum amount of silk in their webs to catch prey. The web has to stop a rapidly flying insect nearly instantly in a manner that allows the prey to become entangled and trapped. To achieve this end, the web must absorb the energy of momentum of the moving insect without breaking. Moreover, the web must also possess mechanical properties that serve to retain the insect. Gosline et al. (8) have reviewed several aspects of this property and concluded that spider silk and the web are nearly optimally designed for each other.
The present inventors have tested major and minor ampullate and egg case silks from both Nephila clavipes and Araneus gemmoides using standard mechanical testing methods (18). The silks were found to exceed the published data for tensile strength by a substantial margin. This was due to the use of the minimum diameter at ten points along the tested fiber for the calculation instead of the average diameter calculated from the density, length and weight. This minimum diameter is about 50% of the average diameter and since silks are likely to break at the narrowest point, these values may be more characteristic of the true properties of these silk fibers. Further examination of spider silk fibers (19) using scanning electron microscopy has confirmed the large variation in diameter of the fibers.
As with any polymer, especially those comprised of protein, there are numerous factors including temperature, hydration state, and rate of extension that can affect tensile strength and elasticity. Despite these caveats, it is clear that dragline silk is a unique biomaterial. Dragline silk can absorb more energy prior to breaking than nearly any commonly used material. It is nearly as strong as several of the current synthetic fibers but can outperform them in many applications where total energy absorption is required.
In 1990, the first spider silk protein from major ampullate silk was cloned in the form of a MaSp 1 cDNA from N. clavipes (23). The led to the appreciation that a second major ampullate silk protein existed which comprised a proline-containing peptide which was absent from the cDNA sequence coding for MaSp 1. This led to the cloning and sequencing of the cDNA for the second major ampullate silk protein, MaSp 2 (10).
The sizes of the mRNA and genes for MaSp1 and MaSp2 have been determined by analysis of Northern blots, restriction digestion patterns, and Southern blots of genomic DNA. The mRNA sizes for MaSp 1 and 2 are approximately 12.5 and 10.5 kb, respectively. The genomic DNA studies all indicate the absence of large introns in the coding regions and the lack of any detectable introns in the main portion of the gene.