In order to manufacture vast quantities of purified spider silk protein for wet spin­ning methodologies, the expression system for protein production needs to be ro­bust, cost-effective and offer rapid biochemical strategies to purify the recombinant fibroins for the spinning process. At least five other natural fibroin cDNA sequences have been used for recombinant expression, which include MaSp2, the tubuliform spidroins TuSp1 and ECP-2, PySp2, and AcSp1 (Fig. 1.6).39’5254a However, despite the availability of the seven different spidroin genetic blueprint sequences, the ma­jority of the recombinant expression studies have focused on dragline silk fibroins, specifically MaSp1. Currently, it is unclear how many companies are deeply in­vested in the large-scale production of spider silk. A few companies with spider silk interest have surfaced in the news, including a San Francisco Bay Area company, Refactored Materials, Inc., as well as a Japanese startup company, named Spiber®. Both have been pursuing the process for large-scale production of synthetic spider silk fibers in either yeast or bacteria, respectively. Spiber® has reported that it can produce several hundreds of grams of recombinant spider silk protein per day.

If tons of spider silk materials are to be manufactured for global distribution, scaling the procedure to synthesize sufficient quantities of fibroins from transgenic

cells or organisms must be optimized. Therefore, identifying the most efficient ex­pression system for recombinant spider protein production has been intensely inves­tigated and still remains as a barrier. So far, it would seem that bacteria or yeast are emerging as the likely candidates. Based upon expression studies, for example, in the methylotrophic yeast P. pastoris, it is reasonable to assume that expression of some recombinant fibroins can achieve 1 g/L of culture.72 Typically, one gram of pu­rified recombinant spidroin could produce about 29,527 feet of silk. To produce one ton of spider silk, which is the equivalent of approximately 998,412 grams, it would require about 100,000 L of culture. This is well within the range of some large in­dustrial sized fermenters that have volume capacities that can exceed 25,000 L. One of the chief advantages for using P. pastoris includes the secretion of the recombi­nant proteins into the extracellular medium, which allows for rapid purification of proteins due to the fact that little, if any, other proteins are secreted into the liquid media. Furthermore, it eliminates the need to analyze the cells and restart cultures for expression, a process that can be tedious, increase production times, and result in higher production costs and manufacturing prices. Additionally, P pastoris can be grown in bioreactors to high densities on mineral salt media and has been shown to be effective for expression of very large and complex proteins, such as collagens, which also require the coexpression of collagen prolyl 4-hydroxylase for the ther­mal stability of collagens.73 As more is revealed about the proteins involved in the spider silk assembly pathway, these components can be integrated into the expres­sion system and presumably lead to better products.

1.4 CONCLUSION

Over the past several years, the spider silk community has advanced their under­standing regarding the protein compositions of the different fiber types. With these advances, many partial cDNAs and several complete genetic blueprints coding for spider silk proteins are available for recombinant protein expression. Additionally, wet-spinning methodologies that use purified, recombinant spider protein as spin­ning dopes are becoming more commonly reported in the literature. Still, the need for further progress to increase the quantities of recombinant proteins manufactured by host cells, along with improvements and new strategies for mechanical spinning will need to be developed if spider silk synthesis is to successfully reach large-scale production with material properties that rival natural silks. Undoubtedly, the differ­ent applications for spider silk proteins as biocomposites seem endless.

1.5 ACKNOWLEGMENT

We thank Drs. Joan and Geoff Lin-Cereghino with expression studies in P pastoris. In addition, we thank Yang Hsia, Eric Gnesa, Felicia Jeffrey, Thanh Phanm, Connie Liu, Christine Ho, Lisa Pham and Ryan Pacheco for their valuable contributions. We also are indebted to Dr. Mark Brunei at the University of the Pacific, Department of Biology, for his assistance with the scanning electron microscope.

KEYWORDS

• Biocomposites

• Mechanical Properties

• Protein

• Spider Silk