Recombinant expression of hydroxylated human collagen in Escherichia coli

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

CO2 adsorption on Aluminosilicate Single-Walled Nanotubes of Imogolite Type

Adsorption of CO2 at subatmospheric pressure at temperatures about ambient has been studied on three materials: (i) imogolite (IMO, chemical formula (OH)3Al2O3SiOH)) a hydrated alumino-silicate occurring as nanotubes (NTs) with bridged AlOHAl groups at the outer surface and Si−OH groups at the inner surface; (ii) an imogolite-like material (Me-IMO, chemical formula (OH)3Al2O3SiCH3) with Si-CH3 groups replacing Si−OH at NTs inner surface; (iii) a material (Me-IMO-NH2) obtained by grafting 3-aminopropylsilane at the outer surface of Me- IMO. All materials, being in the form of NTs, exhibit rather high specific surface area values (355−665 m2 g−1) and are accessible to CO2 molecules. Infrared spectroscopy shows that carbon dioxide may interact in a variety of ways. At the inner surface of IMO, linear molecular species are reversibly formed by interaction with silanols, whereas at the outer surface carbonate-like species are given rise with partial reversible character. With Me-IMO, no interaction takes place at the inner surface: linear species are formed in the intertube nanopores as well as carbonate species as in the case of IMO. Finally, with Me-IMO-NH2, all species present in Me-IMO are found, as well as reversible carbamate species arising from the reaction with amino groups. Optical isotherms concerning molecular adsorption have Langmuir character, whereas those for the reversible formation of carbonates/carbamates are of Henry-type. Volumetric isotherms are interpreted as due to two independent families of adsorption sites, respectively Langmuir and Henry: comparison between optical isotherms (measured at ca. 33 °C) and volumetric isotherms (measured at 0 °C) allows a semiquantitative estimate of the adsorption enthalpy for molecular species, corresponding to ca. −20 kJ mol−1, for linear species reversibly formed by interaction with inner silanols in IMO, and to a relatively high adsorption enthalpy for molecular species formed in the larger intertube nanopores of Me-IMO (ca. −32 kJ mol−1)