Biomaterial Applications of Recombinant Bacterial Collagens

描述（由申请人提供）：对于生物医学和生物材料应用，需要易于序列修饰和大规模生产水平的定义明确的标准化胶原蛋白。本研究的目的是在大肠杆菌中高效表达重组细菌胶原蛋白。大肠杆菌中使用作为基础科学工具，用于表征生物活性胶原结构域，并作为支架，以促进骨生成的人类干细胞。从S.化脓菌和其它四种细菌的高产量已经显示出尽管不存在羟脯氨酸，但仍形成高稳定性的三螺旋分子。将表征由细菌胶原蛋白形成的更高级结构聚集体，并交联以获得稳定性，并表征所形成材料的机械性能。胶原蛋白变体还将被设计为含有与细菌胶原蛋白样结构域相邻的微调卷曲螺旋，以扩大增加分子稳定性的机会，形成异源三聚体分子以促进蛋白质缔合，并包含特异性细胞结合和基质金属蛋白酶（MMP）切割位点，以更好地定义胶原蛋白细胞受体并控制蛋白质的周转。将研究定制的细菌胶原蛋白支持人骨髓源性间充质干细胞作为骨替代的潜在生物材料的生长和分化的能力。使用一系列成骨表型和基因型标记物，将具有和不具有插入生物信号的细菌胶原蛋白与提取的哺乳动物胶原蛋白进行比较。此外，将使用巨噬细胞和树突状细胞筛选以及小鼠皮下植入物在体外和体内将对各种胶原的炎症反应与动物胶原进行比较。这些比较将提供细胞、组织和动物水平上对胶原蛋白变体的生物学反应的基线数据。在这个系统中的高产率和简单的遗传操作将允许不同的生物活性位点的组合，并允许交互式优化，以设计适当的结构和生物学特性的生物材料，微调干细胞反应相关的基本细胞生物学和应用的生物材料和组织工程的需要。跨学科的合作者团队带来了重要的互补专业知识，使该项目取得成功，Brodsky博士在胶原蛋白结构方面的广泛工作，Kaplan博士在干细胞和生物材料方面的记录，Ramshaw博士在胶原蛋白，生物材料和产品开发方面的经验，以及我们的顾问在基质金属蛋白酶（Nagase博士）和DDR受体（Leitinger博士）方面的专业知识。 公共卫生相关性：对于生物医学和生物材料应用，需要易于序列修饰和大规模生产的定义明确的标准化胶原蛋白。细菌胶原蛋白系统的发展，包括生物活性位点，并形成明确的层次结构，将彻底改变这种重要的蛋白质在基础和应用研究中的使用。

DESCRIPTION (provided by applicant): There is a need for well-defined standardized collagen proteins amenable to easy sequence modifications and large scale production levels for biomedical and biomaterial applications. This proposal aims to express recombinant bacterial collagens in high yield in E. coli to use both as a basic science tool for characterizing biologically active collagen domains and as a scaffold to promote bone generation by human stem cells. Bacterial collagen proteins expressed from S. pyogenes and four other bacteria in high yield have been shown to form triple-helical molecules of high stability despite the absence of hydroxyproline. Higher order structural aggregates formed by bacterial collagens will be characterized and cross-linked for stability, and the mechanical properties of materials formed will be characterized. Collagen variants will also be designed to contain a trimmer coiled coil adjacent to the bacterial collagen-like domain to expand the opportunities for increased molecular stability, formation of heterotrimeric molecules to foster protein association, and the inclusion of specific cell binding and matrix metalloproteinase (MMP) cleavage sites in order to better define collagen cell receptors and control turnover of the protein. The ability of tailored bacterial collagen proteins to support growth and differentiation of human bone marrow derived mesenchymal stem cells as potential biomaterials for bone replacement will be investigated. Bacterial collagens with and without inserted biological signals will be compared with extracted mammalian collagens using a range of osteogenic phenotypic and genotypic markers. In addition, inflammatory responses to the various collagens will be compared in vitro and in vivo to animal collagens using macrophage and dendritic cell screens, and subcutaneous implants in mice. These comparisons will provide baseline data on biological responses at the cell, tissue and animal level to the collagen variants. High yields and straightforward genetic manipulations in this system will allow combinations of different biologically active sites and permit interactive optimization to design biomaterials of appropriate structural and biological properties to fine tune stem cell responses related to basic cell biology and applied biomaterials and tissue engineering needs. The interdisciplinary team of collaborators brings important complementary expertise to make this project successful, with Dr. Brodsky's extensive work on collagen structure, Dr. Kaplan's track record with stem cells and biomaterials, Dr. Ramshaw's experience with collagen, biomaterials and product development, and our consultants expertise in matrix metalloproteinases (Dr. Nagase) and DDR receptors (Dr. Leitinger). PUBLIC HEALTH RELEVANCE: There is a need for well-defined standardized collagen proteins amenable to easy sequence modifications and large scale production for biomedical and biomaterial applications. The development of a bacterial collagen system to incorporate biologically active sites and to form well-defined hierarchical structures would revolutionize the use of this important protein in both fundamental and applied studies.