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rdf:type |
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lifeskim:mentions |
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pubmed:dateCreated |
2001-1-17
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pubmed:abstractText |
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.
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pubmed:commentsCorrections |
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-10428813,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1323838,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1397333,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1607009,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1622419,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1703407,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-1860888,
http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-2440339,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-3133372,
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http://linkedlifedata.com/resource/pubmed/commentcorrection/11104702-9920912
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pubmed:language |
eng
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pubmed:journal |
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pubmed:citationSubset |
IM
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pubmed:chemical |
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pubmed:status |
MEDLINE
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pubmed:month |
Dec
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pubmed:issn |
0264-6021
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pubmed:author |
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pubmed:issnType |
Print
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pubmed:day |
15
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pubmed:volume |
352 Pt 3
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
907-11
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pubmed:dateRevised |
2009-11-18
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pubmed:meshHeading |
pubmed-meshheading:11104702-Animals,
pubmed-meshheading:11104702-Cell Line,
pubmed-meshheading:11104702-Chickens,
pubmed-meshheading:11104702-Chymotrypsin,
pubmed-meshheading:11104702-Circular Dichroism,
pubmed-meshheading:11104702-Collagen,
pubmed-meshheading:11104702-Gene Expression,
pubmed-meshheading:11104702-Humans,
pubmed-meshheading:11104702-Hydroxylation,
pubmed-meshheading:11104702-Hydroxyproline,
pubmed-meshheading:11104702-Molecular Weight,
pubmed-meshheading:11104702-Peptide Fragments,
pubmed-meshheading:11104702-Procollagen-Proline Dioxygenase,
pubmed-meshheading:11104702-Protein Structure, Secondary,
pubmed-meshheading:11104702-Protein Subunits,
pubmed-meshheading:11104702-RNA, Messenger,
pubmed-meshheading:11104702-Recombinant Proteins,
pubmed-meshheading:11104702-Temperature,
pubmed-meshheading:11104702-Thermodynamics,
pubmed-meshheading:11104702-Transfection,
pubmed-meshheading:11104702-Trypsin
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pubmed:year |
2000
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pubmed:articleTitle |
Coexpression of alpha and beta subunits of prolyl 4-hydroxylase stabilizes the triple helix of recombinant human type X collagen.
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pubmed:affiliation |
Department of Experimental Medicine I, Nikolaus-Fiebiger Center für Molecular Medicine, University of Erlangen-Nuremberg, Glückstrasse 6, D-91054 Erlangen, Germany.
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pubmed:publicationType |
Journal Article,
Research Support, Non-U.S. Gov't
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