Expressed Bacterial Triple-Helical Products as Tissue Engineering Scaffolds

描述（由申请人提供）：胶原蛋白三螺旋基序的独特性质包括其分子流体动力学性质，广泛的水合作用，结合多种配体的能力，以及自结合形成原纤维和其他高阶结构的能力。这些独特的特征已经被大自然利用来填补广泛的结构和功能空缺，并被科学家设计出许多生物材料和生物医学用途的重要产品。由于提取胶原的使用存在问题和担忧，人们的注意力已经转移到重组动物胶原的生产上，但由于翻译后修饰的问题，高效的生产已经放缓。与其关注特定类型的动物胶原，我们建议关注基序的内在特性，使用细菌三螺旋模块，它具有高稳定性，没有翻译后修饰。基于细菌“胶原样”序列的稳定三螺旋模块将用于在高产大肠杆菌冷休克表达系统中形成三螺旋聚合物。重复模块将用于获得与动物纤维形成胶原相当的长度，我们假设这些三螺旋蛋白将自组装形成纤维结构。已知相互作用的确定的人胶原蛋白序列将插入相邻的细菌胶原蛋白模块之间，以产生具有功能结合位点的嵌合体。在细菌三螺旋模块之间的重复（Gly-X-Y）n序列中引入基膜型断裂将用于创建更接近基膜胶原网络的柔性或扭结型结构，并可能为干细胞培养提供底物。这种新颖的方法计划捕捉胶原蛋白三螺旋基序的灵活性和优势，同时保留细菌表达系统的简单性。该系统的产物将是理想的操作和应用作为支架在组织工程。摘要：一种获得重复细菌胶原样序列的高效生产的新方法计划在保留细菌表达系统的简单性的同时捕获胶原三螺旋motrf的灵活性和优势。该系统的产物将是理想的操作和应用作为支架在组织工程。

DESCRIPTION (provided by applicant): The unique properties of the collagen triple-helix motif include its molecular hydrodynamic properties, extensive hydration, ability to bind diverse ligands, and the capacity self-associate to form fibrils and other higher order structures. These distinctive features have been exploited by nature to fill a wide range of structural and functional niches, and by scientists to design numerous products important for biomaterial and biomedical uses. Because of problems and concerns with the use of extracted collagens, attention has shifted to production of recombinant animal collagens, but efficient production has been slowed down addressing issues of post-translational modification. Rather than focus on specific types of animal collagens, we propose to focus on the intrinsic properties of the motif, using a bacterial triple-helix module which has high stability with no post-translational modifications. Stable triple-helix modules based on bacterial "collagen-like" sequences will be utilized to form triple-helical polymers in a high yield E. coli cold shock expression system. Repeating modules will be used to attain lengths comparable to that of animal fibril forming collagens, and we hypothesize that these triple-helical proteins will self-assemble to form fibrillar structures. Defined human collagen sequences involved in known interactions will be inserted between adjacent bacterial collagen modules to produce chimeras with functional binding sites. Introduction of basement membrane type breaks in the repeating (Gly-X-Y)n sequence between bacterial triple-helix modules will used to create a flexible or kinked type structure that more closely models basement membrane collagen networks and may provide a substrate for stem cell cultures. This novel approach plans to capture the flexibility and advantages of the collagen triple-helix motif while retaining the simplicity of a bacterial expression system. The products of this system will be ideal for manipulations and applications as scaffolds in tissue engineering. Lay Summary: A novel approach to gain efficient production of repeating bacterial collagen-like sequences plans to capture the flexibility and advantages of the collagen triple-helix motrf while retaining the simplicity of a bacterial expression system. The products of this system will be ideal for manipulations and applications as scaffolds in tissue engineering.