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. ? ? ?
