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

Klaus Wagner, Ernst Pöschl, Javier Turnay, Jeong-Mi Baik, Taina Pihlajaniemi, Svenja Frischholz, Klaus von der Mark

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15 Citations (Scopus)

Abstract

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 degrees C. By comparison, native chicken type X procollagen has a T(m) of 46 degrees C. 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 proalpha1(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 degrees C 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 degrees C, confirming the presence of a triple helix. The same T(m) of 41 degrees C 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.

Original languageEnglish
Pages (from-to)907-911
Number of pages5
JournalBiochemical Journal
Volume352
Issue number3
DOIs
Publication statusPublished - 15 Dec 2000

Keywords

  • Animals
  • Cell Line
  • Chickens
  • Chymotrypsin
  • Circular Dichroism
  • Collagen
  • Gene Expression
  • Humans
  • Hydroxylation
  • Hydroxyproline
  • Molecular Weight
  • Peptide Fragments
  • Procollagen-Proline Dioxygenase
  • Protein Structure, Secondary
  • Protein Subunits
  • RNA, Messenger
  • Recombinant Proteins
  • Temperature
  • Thermodynamics
  • Transfection
  • Trypsin

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