Scalable Purification of Silk-Like Proteins from Transgenic Tobacco

从转基因烟草中大规模纯化丝状蛋白

• 批准号: 7253828
• 负责人: William Richard Marcotte
• 金额: $ 22.05万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7253828
• 关键词: Absorbable Implants; Address; Affinity; Affinity Chromatography; Agriculture; Agrobacterium; Behavior; Binding; Bombyx; Bone and Cartilage Funding; C-terminal; Cells; Cellulose; Characteristics; Consensus; Coupling; Data; Development; Elasticity; Excision; Exhibits; Farming environment; Fiber; Gene Expression; Genes; Harvest; Immunoblotting; Kilogram; Ligaments; Marketing; Methods; N-terminal; Numbers; Performance; Plant Proteins; Plants; Polymerase Chain Reaction; Procedures; Process; Production; Proteins; Recombinant Proteins; Recombinants; Research; Role; Silk; Spiders; Standards of Weights and Measures; Surgical sutures; System; Techniques; Tendon structure; Tensile Strength; Textiles; Tissue Engineering; Tissues; Tobacco; Transgenic Organisms; Transgenic Plants; base; bioprocess; commercial application; cost; cost effective; improved; intein; novel strategies; protein expression; protein purification; resilience; scaffold; scale up; spidroin 1; spidroin 2

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).

描述(申请人提供)：蜘蛛丝是一种蛋白质纤维，表现出抗拉强度、弹性和弹性的非凡组合。这使得它们成为生物医学应用的理想选择，例如人工肌腱和韧带，骨和软骨植入的细胞支架，以及可生物降解的微缝合。与养蚕不同，蜘蛛的养殖是不现实的，因为产丝量低，而且有领地行为。因此，几个研究小组试图在各种转基因表达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。高度重复的丝素基因在植物中是稳定的，积累到可察觉的水平，植物表达允许使用标准农业实践进行几乎无限的放大。然而，植物丝绸生产的一个主要障碍是需要一种可扩展且成本效益高的方法来从大量植物组织中提纯丝蛋白。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域的蜘蛛丝蛋白。纤维素结合结构域(CBD)将被用作亲和标签，用于在廉价的纤维素基质上纯化蚕丝蛋白。一些CBD-Silk构建物还将整合内含子结构域，用于亲和纯化后自动催化去除CBD。我们的蜘蛛丝基因将优于以前研究中使用的那些，因为：1)大量的蜘蛛蛋白共识重复，2)包含保守的N-端和C-端序列，3)加入CBD亲和标签以改善下游加工。值得注意的是，我们的丝绸基因的N-末端蜘蛛蛋白序列从未包括在重组表达的丝绸中，因为这些序列直到最近才被确定。我们的初步数据支持我们的假设，即蜘蛛丝可以在烟草中高效表达，CBD-丝可以通过纤维素亲和力得到纯化。通过结合利用烟草高效表达的巨大潜力和经济上可扩展的CBD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。这项提议的重点是开发具有成本效益的提纯程序(生物处理)的扩大。

项目成果

Recombinant Dragline Silk-Like Proteins-Expression and Purification.

重组拉铲丝状蛋白的表达和纯化。

• 发表时间: 2011
• 期刊: AATCC review
• 作者: Gaines,WilliamA;MarcotteJr,WilliamR
• 通讯作者: MarcotteJr,WilliamR

Identification and characterization of multiple Spidroin 1 genes encoding major ampullate silk proteins in Nephila clavipes.

• DOI: 10.1111/j.1365-2583.2008.00828.x
• 发表时间: 2008-09
• 期刊: Insect molecular biology
• 影响因子: 2.6
• 作者: Gaines WA 4th;Marcotte WR Jr
• 通讯作者: Marcotte WR Jr

NMR assignments of the N-terminal domain of Nephila clavipes spidroin 1.

Nephila clavipes spidroin 1 N 末端结构域的 NMR 分配。

• DOI: 10.1007/s12104-010-9284-z
• 发表时间: 2011
• 期刊: Biomolecular NMR assignments
• 影响因子: 0.9
• 作者: Parnham,Stuart;Gaines,WilliamA;Duggan,BrendanM;MarcotteJr,WilliamR;Hennig,Mirko
• 通讯作者: Hennig,Mirko