Engineering Cell Interaction Sites in Collagen-Mimetic Biopolymers

The ability to fabricate full-length, collagen-mimetic biopolymers of specified sequence has been elusive, primarily due to difficulties related to gene synthesis and polymer stability.  We have created a platform which enables us to fabricate de novo collagen with prescribed sequence.  These polymers are encoded by modular genes, the assembly of which required overcoming the challenges of mishybridization during PCR assembly caused by the repeating Gly-X-Y sequence that is typical for collagen.  The genes are then introduced into a genetically-modified Saccharomyces cerevisiae expression system which has been optimized to produce sufficiently high proline hydroxylation levels for thermally-stable collagen.  Circular dichroism and atomic force microscopy both showed triple helical structure that is characteristic of native collagen.  We demonstrate the incorporation of non-native alpha2-beta1 integrin binding sites of defined sequence, frequency, and specific location within one polymeric strand, and we investigate the effects of changing these variables within the polymer on mammalian cell response.  The capability to produce collagen-mimetic variants enables the manipulation of the cellular microenvironment for applications such as regenerative medicine, drug delivery, and fundamental studies of the extracellular matrix.