Coexpression of α and β subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen

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 (T-m) of 31 degreesC. By comparison, native chicken type X procollagen has a T-m of 46 degreesC. To stabilize the triple helix of hrColX, an hrColX-expressing clone (A6/16) was co-transfected with both alpha and beta subunits of human prolyl 4-hydroxylase. Clones were selected that secreted pro alpha1(X) collagen chains with an apparent molecular mass of 75 kDa and an increased hydroxyproline-to-proline ratio of close to 0.5. As a result of enhanced prolyl hydroxylation, the T-m of the hrColX was increased to 41 degreesC as measured by CD analysis at various temperatures. The CD spectra indicated a minimum ellipticity at 198 nm and a peak at 225 nm at 20 degreesC, confirming the presence of a triple helix. The same T-m of 41 degreesC was measured for the triple-helical core fragments of hrColX of 60-65 kDa 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.