title: 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 that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cerevisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.
authors.affiliation: Department of Chemical Engineering and Materials Science, University of California, Irvine, California 92697, USA.
authors.forename: Sam Wei Polly
authors.initials: SW
authors.lastname: Chan
authors.forename: She-Pin
authors.initials: SP
authors.lastname: Hung
authors.forename: Senthil Kumar
authors.initials: SK
authors.lastname: Raman
authors.forename: G Wesley
authors.initials: GW
authors.lastname: Hatfield
authors.forename: Richard H
authors.initials: RH
authors.lastname: Lathrop
authors.forename: Nancy A
authors.initials: NA
authors.lastname: Da Silva
authors.forename: Szu-Wen
authors.initials: SW
authors.lastname: Wang
mesh_terms: D040761:Biomimetic Materials / Q000737:chemistry*; D003001:Cloning, Molecular; D024061:Collagen Type III / Q000737:chemistry / Q000235:genetics*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D006801:Humans; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D010957:Plasmids; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
chemical_list: D024061:Collagen Type III; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D003545:Cysteine