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

Wagner, Klaus, Pöschl, Ernst, Turnay, Javier, Baik, Jeong-Mi, Pihlajaniemi, Taina, Frischholz, Svenja and von der Mark, Klaus (2000) Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen. Biochemical Journal, 352 (3). pp. 907-911. ISSN 0264-6021

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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.

Item Type:	Article
Uncontrolled 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
Faculty \ School:	Faculty of Science > School of Biological Sciences
UEA Research Groups:	Faculty of Science > Research Groups > Cells and Tissues
Depositing User:	Pure Connector
Date Deposited:	13 Jan 2016 16:00
Last Modified:	19 Apr 2023 23:45
URI:	https://ueaeprints.uea.ac.uk/id/eprint/56303
DOI:	10.1042/bj3520907

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