Recombinant Expression of Human Type 1 Collagen for Tissue Engineering and Biomaterials

摘要 胶原蛋白形成高度组织化的三维网络，促进细胞附着、迁移， 增殖和分化。强度、持久性和生物相容性是 胶原蛋白，使他们非常适合作为支架在组织工程的努力，修复和重建 组织、骨骼和皮肤。目前，这些胶原蛋白的主要来源是动物来源的，这导致 问题（可靠性、安全性、不一致性等）。虽然重组生产人类胶原蛋白有望 最可能的解决方案是，现有技术的蛋白质生产技术在生产蛋白质中效率低下， 全长功能性胶原蛋白因此，对重组蛋白的需求 胶原及其供应。出处设计了一个独特的战略，以克服的局限性， 这些重组蛋白生产系统。我们建议使用我们的新平台来有效地合成 全长人I型胶原蛋白，具有高滴度的所需翻译后修饰。我们将 证明重组人I型胶原类似于其天然对应物。我们建议生成 表达全长人I型胶原的高产菌株，其显示适当的脯氨酰 羟基化，并优化其在不同规模（摇瓶，生物反应器）的生产工艺。 最后，这里描述的方法应该导致重组蛋白的成本有效的生产策略。 人I型胶原蛋白，并减轻这种胶原蛋白用于组织的可用性的严重限制 工程和作为生物材料在其他生物医学应用。此外，廉价的 人类I型胶原蛋白的来源应该允许研究人员扩大胶原蛋白作为生物材料的作用， 组织工程应用成功完成本第一阶段提案中提出的工作， 开辟了定制胶原蛋白特性的途径，这是一个赋予新功能的长期目标， 反过来可以导致治疗和生物医学应用中的新应用。

Abstract Collagen forms a highly organized, three-dimensional network that facilitates cell attachment, migration, proliferation and differentiation. Strength, persistence and biocompatibility are the inherent properties of collagens that make them well suited as scaffolds in tissue engineering efforts for repair and reconstruction of tissue, bone, and skin. Currently, the primary source of these collagens is animal derived, which causes concerns (reliability, safety, inconsistency, etc). While recombinant production of human collagens promises the most likely solution, the state of art protein production technologies suffer inefficiencies in the production of full length, functional collagens. As such, there’s a recognizable gap between the demand for recombinant collagens and the supply thereof. Provenance has devised a unique strategy for overcoming the limitations of these recombinant protein production systems. We propose to use our novel platform to efficiently synthesize full length human type I collagens with the required post translational modifications at high titers. We will demonstrate the recombinant human type I collagen resembles its native counterpart. We propose to generate highly productive strains expressing full length human type I collagen, that shows the appropriate prolyl hydroxylation, and optimize their production processes at different scales (shake flasks, bioreactors). Ultimately, the approach described here should lead to a cost effective production strategy for recombinant human type I collagen, and alleviate the severe limitations in the availability of this collagen for tissue engineering, and as biomaterials in other biomedical applications. In addition, the availability of an inexpensive source of human type I collagen should allow researchers to expand the role of collagen as a biomaterial for tissue engineering applications. Successful completion of the work proposed in this Phase I proposal can open avenues towards tailoring properties of collagens, a long term goal to impart new functionalities which in turn can lead to novel applications in therapeutic and biomedical applications.