title: Recombinant expression of hydroxylated human collagen in Escherichia coli.
abstract: Collagen is the most abundant protein in the human body and thereby a structural protein of considerable biotechnological interest. The complex maturation process of collagen, including essential post-translational modifications such as prolyl and lysyl hydroxylation, has precluded large-scale production of recombinant collagen featuring the biophysical properties of endogenous collagen. The characterization of new prolyl and lysyl hydroxylase genes encoded by the giant virus mimivirus reveals a method for production of hydroxylated collagen. The coexpression of a human collagen type III construct together with mimivirus prolyl and lysyl hydroxylases in Escherichia coli yielded up to 90 mg of hydroxylated collagen per liter culture. The respective levels of prolyl and lysyl hydroxylation reaching 25 % and 26 % were similar to the hydroxylation levels of native human collagen type III. The distribution of hydroxyproline and hydroxylysine along recombinant collagen was also similar to that of native collagen as determined by mass spectrometric analysis of tryptic peptides. The triple helix signature of recombinant hydroxylated collagen was confirmed by circular dichroism, which also showed that hydroxylation increased the thermal stability of the recombinant collagen construct. Recombinant hydroxylated collagen produced in E. coli supported the growth of human umbilical endothelial cells, underlining the biocompatibility of the recombinant protein as extracellular matrix. The high yield of recombinant protein expression and the extensive level of prolyl and lysyl hydroxylation achieved indicate that recombinant hydroxylated collagen can be produced at large scale for biomaterials engineering in the context of biomedical applications.
authors.affiliation: Institute of Physiology, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Rutschmann
authors.forename: Stephan
authors.initials: S
authors.lastname: Baumann
authors.forename: Jürg
authors.initials: J
authors.lastname: Cabalzar
authors.forename: Kelvin B
authors.initials: KB
authors.lastname: Luther
authors.forename: Thierry
authors.initials: T
authors.lastname: Hennet
mesh_terms: D024061:Collagen Type III / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D013058:Mass Spectrometry; D060847:Metabolic Engineering / Q000379:methods*; D056686:Mimiviridae / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase / Q000235:genetics / Q000378:metabolism; D064828:Prolyl Hydroxylases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D024061:Collagen Type III; D011994:Recombinant Proteins; D064828:Prolyl Hydroxylases; D008240:Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase