We have reported previously on the expression of recombinant human type X collagen (hrColX) in HEK 293 and HT 1080 cells by using the eukaryotic expression vector pCMVsis (in which CMV stands for cytomegalovirus). Several stably transfected clones secreted full-length triple-helical hrColX molecules in large amounts, but the secreted collagen was underhydroxylated, with a hydroxyproline-to-proline ratio of 0.25 and a melting temperature (Tm) of 31°C. By comparison, native chicken type X procollagen has a Tm of 46°C. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both α and β subunits of human prolyl 4-hydroxylase. Clones were selected that secreted proα1(X) collagen chains with an apparent molecular mass of 75kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the Tm of the hrColX was increased to 41°C as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198nm and a peak at 225nm at 20°C, confirming the presence of a triple helix. The same Tm of 41°C was measured for the triple-helical core fragments of hrColX of 60Ő65kDa that were retained after brief digestion with chymotrypsin/trypsin at increasing temperatures. This shows that the human cell line HEK-293 is suitable for the simultaneous expression of three genes and the stable production of substantial amounts of recombinant, fully hydroxylated type X collagen over several years.

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