Saturday, May 18, 2024
5/18/2024
DESCRIPTION (provided by applicant): Spider silks are protein fibers that exhibit a remarkable combination of tensile strength, resilience, and elasticity. This makes them desirable for biomedical applications such as artificial tendons and ligaments, cell scaffolds for bone and cartilage implants, and biodegradable micro-sutures. Unlike silkworms, farming of spiders is unrealistic because of low silk yields and territorial behavior. Thus, several research groups have attempted to produce spider silk genes in a variety of transgenic expression hosts. While most expression systems have shown significant problems, plant expression systems are very promising. The highly repetitive silk genes are stable in plants, accumulate to appreciable levels and plant expression allows a nearly unlimited scale-up using standard agricultural practices. However, one major hurtle for plant-based silk production is the need for a scalable and cost-effective method to purify silk protein from bulk quantities of plant tissue. To address this problem, we will express spider silk proteins fused to cellulose-binding domains in transgenic tobacco. The cellulose-binding domain (CBD) will be used as an affinity tag for purification of silk protein on an inexpensive cellulose matrix. Some CBD-silk constructs will also incorporate an intein domain for autocatalytic removal of the CBD after affinity purification. Our spider silk genes will be superior to those used in previous studies due to: 1) a large number of spidroin consensus repeats, 2) inclusion of conserved N-terminal and C-terminal sequences, 3) incorporation of CBD affinity tag for improved downstream processing. Notably, the N-terminal spidroin sequences of our silk genes have never been included in a recombinantly expressed silk because these sequences have only recently been determined. Our hypothesis that spider silk can be efficiently expressed in tobacco and that CBD-silk can be purified by cellulose affinity is supported by our preliminary data. By coupling the ability to exploit the immense iomass possibilities of high-level tobacco expression and the efficiency of economically-scalable CBD-based purification, this study aims to develop a feasible method for large-scale production of spider silk protein. One of the most significant challenges for the production of recombinant materials in a transgenic system is the scalability of the purification process. This proposal is focused on the development of cost-effective scale-up for purification procedures (bioprocessing).
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