Recombinant human collagen and biomimetic variants using a de novo gene optimized for modular assembly.
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.
Chan
Sam Wei Polly
SW
Department of Chemical Engineering and Materials Science, University of California, Irvine, California 92697, USA.
Hung
She-Pin
SP
Raman
Senthil Kumar
SK
Hatfield
G Wesley
GW
Lathrop
Richard H
RH
Da Silva
Nancy A
NA
Wang
Szu-Wen
SW
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
D024061:Collagen Type III; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D011393:Procollagen-Proline Dioxygenase; D003545:Cysteine