(694a) Design of Recombinant Human Collagen and Collagen-Based Variants

Collagen, the most abundant extracellular matrix protein, demonstrates a dynamic relationship with its co-residing cells. Polypeptide regions within its native sequence have been shown to modulate cellular behavior. Therefore, the ability to create a novel class of collagen-mimetic polymers in which these functional regions are manipulated would be significant for applications in regenerative medicine. Despite this potential, however, the de novo synthesis of full-length collagen has been prevented by the characteristic (Glycine-X-Y)_n sequence repetition in the triple-helical domain, which adds an extra degree of complexity to the gene synthesis. Using a computational algorithm to optimize oligonucleotide hybridization, we have successfully created a modular gene encoding full-length native human collagen III. Our modular approach enables flexibility to fabricate collagen variants which were not previously possible. We designed genes encoding collagen-mimetic biopolymers with non-native cell interaction sites and chemical linkage sites in precisely-defined locations. We then expressed these proteins in Saccharomyces cerevisiae and purified the recombinant collagen products. Structural analysis has included atomic force microscopy, and using gel electrophoresis we show that these biopolymer variants are assembled into trimers, as expected. We also demonstrate that these biomolecules are able to support cellular adhesion and growth.