Scalable Purification of Silk-Like Proteins from Transgenic Tobacco

描述（由申请人提供）：蜘蛛丝是蛋白质纤维，具有显著的抗拉强度、回弹性和弹性。这使得它们成为生物医学应用的理想材料，如人造肌腱和韧带，骨和软骨植入物的细胞支架，以及可生物降解的微缝合线。与蚕不同，饲养蜘蛛是不现实的，因为蜘蛛的产丝量低，而且有领地行为。因此，几个研究小组已经尝试在各种转基因宿主中产生蜘蛛丝基因。虽然大多数表达系统都显示出明显的问题，但植物表达系统是非常有前途的。高度重复的丝基因在植物中是稳定的，积累到可观的水平，并且植物表达允许使用标准农业实践几乎无限地扩大规模。然而，以植物为基础的蚕丝生产的一个主要障碍是需要一种可扩展且经济有效的方法来从大量植物组织中纯化蚕丝蛋白。为了解决这个问题，我们将在转基因烟草中表达融合到纤维素结合结构域的蜘蛛丝蛋白。纤维素结合结构域（CBD）将被用作在廉价的纤维素基质上纯化蚕丝蛋白的亲和标签。一些CBD-丝绸结构也将纳入一个内部结构域，用于亲和纯化后的CBD自催化去除。我们的蜘蛛丝基因将优于以往的研究，因为：1)大量的蜘蛛丝共识重复，2)包含保守的n端和c端序列，3)结合CBD亲和标签，以改善下游加工。值得注意的是，我们的蛛丝基因的n端蜘蛛蛋白序列从未被包含在重组表达的蛛丝中，因为这些序列是最近才确定的。我们的假设是蜘蛛丝可以有效地在烟草中表达，并且cbd丝可以通过纤维素亲和纯化得到支持。通过利用烟草高水平表达的巨大质量可能性和经济上可扩展的基于cbd的纯化效率，本研究旨在开发一种大规模生产蜘蛛丝蛋白的可行方法。在转基因系统中生产重组材料的最大挑战之一是纯化过程的可扩展性。该提案的重点是发展具有成本效益的净化程序（生物处理）。

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).