Engineering of collagenous proteins and protein hydrogels for biomedical applications [electronic resource]
Keywords: Bioengineering.

Recombinant proteins have been used widely in both basic research and biomedical applications including protein therapeutics and biomaterials. Many efforts have been devoted to the investigation of novel synthetic strategies for producing recombinant proteins for various applications, which is the focus of this dissertation. In the first section, a biosynthetic strategy was developed to produce collagenous proteins with post-translational modifications in E. coli. Collagen is the most abundant protein in human, and plays a dominant role in maintaining the biological and structural integrity. Recombinant expression of collagens and fragments of collagens is often difficult as their stability requires appropriate proline hydroxylation. Prolyl 4-hydroxylases (P4H) are ascorbate-dependent oxygenases that play key roles in collagen folding by catalyzing the post-translational hydroxylation of specific proline residues on target proteins to form (2S, 4R)-4-hydroxyproline. Thus far, the study of these post-translational modifications has been limited by the lack of a prokaryotic recombinant expression system for producing hydroxylated proteins. Unlike eukaryotic cells such as yeast and insect cells, bacterial cytoplasm cannot activate P4H, which requires an ascorbate co-factor that bacteria do not produce. By introducing a biosynthetic shunt to produce ascorbate-like molecules in E. coli cells that heterologously express human P4H, we have created a strain of E. coli that produces collagenous proteins with (2S, 4R)-4-hydroxyproline. Different levels of proline hydroxylation can be obtained by tuning culture conditions. We have verified that hydroxylation of collagenous materials produced in the new system leads to an increase in thermostability. Using this new system, we have observed hydroxylation patterns indicative of a processive catalytic mode for P4H that is active even in the absence of ascorbate. Our results provide insights into P4H enzymology, and create a foundation for better understanding how post-translational hydroxylation affects proteins. Further, we applied the novel E. coli expression system to produce a collagenous protein, adiponectin, which has many beneficial effects on obesity-related metabolic and cardiovascular disorders, and reverses insulin insensitivity. By introducing key post-translational modification enzymes to E. coli, we have endowed the expression system with capabilities of making necessary modifications on adiponectin for its correct assembly, and thus obtained biomimetic adiponectins. High-molecular weight (HMW) multimers of adiponectin have been obtained, and their biological activities of suppressing endothelial cell apoptosis have been confirmed with in vitro cell assays. In section 2, modular protein polymers have been created through genetic engineering and enzymatically crosslinked into hydrogels with tunable properties. The many challenges currently faced in regenerative medicine research require the development of new, modular biomaterial systems that can serve as scaffolds for cellular maintenance, expansion and growth, and which can be tuned by the user to mimic any necessary aspects of natural ECM to an optimal degree. Toward this goal, we have created a family of block co-polypeptides comprising amino acid sequence elements that allow mild enzymatic crosslinking into gels. These new families of protein polymers were designed to be linear, random coil, and contain either lysine or glutamine, which have the recognition substrates for transglutaminase (TG) crosslinking, evenly spaced along the protein backbone. Crosslinking occurred within two minutes upon the addition of TG under physiological conditions, as determined by particle tracking microrheology. The material properties of the gel can be tuned with the hydrogel composition to mimic cellular microenvironment of different tissues. Furthermore, in order to introduce biofunctionalities into the hydrogels, a versatile expression vector has been engineered that allows the insertion of bioactive protein domains into these block co-polypeptide. For example, a cell adhesion signal based on the RGD sequence from human fibronectin was incorporated. The RGD-containing hydrogel was successful in enhancing cell adhesion, and were also proven to be compatible with the culture of mesenchymal stem cells. The specific nature of these protein polymer precursors of the modular hydrogel composition allows tailoring of mechanical and biochemical properties, rendering these gels valuable for various tissue engineering applications.