title: Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen.
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.
authors: Department of Experimental Medicine I, Nikolaus-Fiebiger Center für Molecular Medicine, University of Erlangen-Nuremberg, Glückstrasse 6, D-91054 Erlangen, Germany.
authors: K
authors: K
authors: Wagner
authors: E
authors: E
authors: Pöschl
authors: J
authors: J
authors: Turnay
authors: J
authors: J
authors: Baik
authors: T
authors: T
authors: Pihlajaniemi
authors: S
authors: S
authors: Frischholz
authors: K
authors: K
authors: von der Mark
mesh_terms: D000818:Animals; D002460:Cell Line; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D003094:Collagen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D006909:Hydroxyproline / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D021122:Protein Subunits; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003094:Collagen; D011393:Procollagen-Proline Dioxygenase; D002918:Chymotrypsin; D014357:Trypsin; D006909:Hydroxyproline