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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB011620-04
Application #
8523854
Study Section
Musculoskeletal Tissue Engineering Study Section (MTE)
Program Officer
Hunziker, Rosemarie
Project Start
2010-09-30
Project End
2014-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
4
Fiscal Year
2013
Total Cost
$300,344
Indirect Cost
$103,418
Name
Tufts University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
073134835
City
Medford
State
MA
Country
United States
Zip Code
02155
An, Bo; Abbonante, Vittorio; Xu, Huifang et al. (2016) Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor. J Biol Chem 291:4343-55
Yigit, Sezin; Yu, Hongtao; An, Bo et al. (2016) Mapping the Effect of Gly Mutations in Collagen on α2β1 Integrin Binding. J Biol Chem 291:19196-207
An, Bo; Lin, Yu-Shan; Brodsky, Barbara (2016) Collagen interactions: Drug design and delivery. Adv Drug Deliv Rev 97:69-84
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Ramshaw, John A M; Werkmeister, Jerome A; Dumsday, Geoff J (2014) Bioengineered collagens: emerging directions for biomedical materials. Bioengineered 5:227-33
Peng, Yong Y; Stoichevska, Violet; Madsen, Soren et al. (2014) A simple cost-effective methodology for large-scale purification of recombinant non-animal collagens. Appl Microbiol Biotechnol 98:1807-15
Peng, Yong Y; Stoichevska, Violet; Howell, Linda et al. (2014) Preparation and characterization of monomers to tetramers of a collagen-like domain from Streptococcus pyogenes. Bioengineered 5:378-85
Peng, Yong Y; Stoichevska, Violet; Schacht, Kristin et al. (2014) Engineering multiple biological functional motifs into a blank collagen-like protein template from Streptococcus pyogenes. J Biomed Mater Res A 102:2189-96
An, Bo; Abbonante, Vittorio; Yigit, Sezin et al. (2014) Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system. J Biol Chem 289:4941-51

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