2010
DOI: 10.1021/bm100052y
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Recombinant Human Collagen and Biomimetic Variants Using a De Novo Gene Optimized for Modular Assembly

Abstract: A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy tha… Show more

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Cited by 19 publications
(51 citation statements)
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References 78 publications
(183 reference statements)
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“…Recombinant collagen has been previously expressed by other groups [12,13] and is commercially available; however, these recombinant systems generate collagen with native sequence and do not demonstrate the ability to design multiple, relatively significant changes at specified locations throughout the biopolymer. This is because a synthetic bottom-up approach has been elusive, due to the difficulties in generating synthetic genes encoding the (glycine-X-Y) N backbone of collagen and the stringent need to post-translationally hydroxylate prolines in the Y-position for stability [14]. Consequently, synthesis of designer collagen-mimetic material has primarily focused on peptides [15e19], unhydroxylated collagen-like polymers from bacteria [20,21], or tandem repeating collagen domains [22].…”
Section: Introductionmentioning
confidence: 99%
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“…Recombinant collagen has been previously expressed by other groups [12,13] and is commercially available; however, these recombinant systems generate collagen with native sequence and do not demonstrate the ability to design multiple, relatively significant changes at specified locations throughout the biopolymer. This is because a synthetic bottom-up approach has been elusive, due to the difficulties in generating synthetic genes encoding the (glycine-X-Y) N backbone of collagen and the stringent need to post-translationally hydroxylate prolines in the Y-position for stability [14]. Consequently, synthesis of designer collagen-mimetic material has primarily focused on peptides [15e19], unhydroxylated collagen-like polymers from bacteria [20,21], or tandem repeating collagen domains [22].…”
Section: Introductionmentioning
confidence: 99%
“…Our research group has developed a platform for creating recombinant human collagen III in which we can specifically tailor the identity, location, and frequency of functional sites within the biopolymer [14,23]. Collagen III was selected as the molecular scaffold because it is a homotrimer; experimentally, this requires only one gene to be synthesized with no need to separate the heterotrimeric populations of product.…”
Section: Introductionmentioning
confidence: 99%
“…Fabrication of Genes Encoding Collagen Variants. Full length collagen-mimetic proteins were fabricated using the 15 The triple-helical region of collagen, for which the gene has been conventionally difficult to assemble, was divided into 12 modules to facilitate synthesis and sequence design. By utilizing the degeneracy of the genetic code, oligonucleotide sequences were optimized to yield higher melting temperatures for correct hybridizations than for incorrect hybridizations.…”
Section: Methodsmentioning
confidence: 99%
“…Primary fragments with and without Cys were mixed-andmatched to assemble secondary fragments with Cys in the desired locations following previously reported protocols; 15 details are provided in Supporting Information. Full length collagen variant genes were assembled from the secondary fragments and sequenced to confirm the correct product.…”
Section: Methodsmentioning
confidence: 99%
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