Collagen, the most abundant extracellular matrix protein, has been widely used in tissue scaffold and drug delivery applications. Our goal is to express full-length human collagen and variants of collagen with modified cellular interaction or structural properties using Saccharomyces cerevisiae as the expression host. Although short synthetic peptides of collagen can be produced, recombinant protein analogous to native full-length collagen has not been synthesized to date. This is due to collagen's relatively long length and its signature repeating Gly-X-Y tripeptide sequence, which results in incorrect oligonucleotide hybridization during gene synthesis. We have constructed, for the first time, a de novo modular collagen gene which encodes for full length human collagen type III. This synthetic gene has been computationally optimized for expression in yeast and to favor correct oligonucleotide hybridization. Modified properties have been introduced by substituting gene modules resulting in a group of collagen variants. The modular nature of the gene makes straightforward fabrication of a wide array of collagen-based biopolymers possible. The collagen and required hydroxylase genes were introduced into yeast using integration and plasmid systems. Gene integrations were confirmed by Southern blot, and hydroxylase and collagen expression were confirmed by Western blot. The recombinant collagen produced was shown to be pepsin-resistant, suggesting triple-helical formation. The strategies we have developed build a solid foundation for producing collagen variants for tailored applications.

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