Expression of human COL1A1 gene in stably transfected HT1080 cells: the production of a thermostable homotrimer of type I collagen in a recombinant system.
A recombinant system was developed for the production of homotrimeric type I collagen in stably transfected HT1080 cells. A DNA construct (COL1A1-CMV) was prepared that contained the cDNA for the human COL1A1 gene under the transcriptional control of the promoter and enhancer of the immediate early gene of CMV. The construct, which also contained a neomycin-resistance gene, was transfected into HT1080 cells, a human fibrosarcoma cell line that synthesizes type IV collagen but does not normally synthesize any of the fibrillar collagens. Cells derived from the neomycin-resistant transfectants were then screened using a polyclonal antibody specific for human pro alpha 1(I) chains in order to identify clones that secreted high levels of the pro alpha(I) chain of type I procollagen. About 2% of neomycin-resistant clones secreted procollagen that consisted of a homotrimer of pro alpha 1(I) chains. The procollagen was post-translationally over-modified as judged by slower migration on SDS-polyacrylamide gel electrophoresis of the pro alpha 1(I) chains compared to pro alpha 1(I) chains of normal type I procollagen. The procollagen was triple helical as assayed by protease digestion with a variable cleavage at 38 degrees C and a thermal transition of both the intact and partially cleaved protein of about 41 degrees C. The system provides a method of expressing genes for fibrillar procollagens so that fully recombinant proteins are generated and easily isolated.
Geddis
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AE
Department of Biochemistry and Molecular Biology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, PA 19107.
Prockop
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D001483:Base Sequence; D003094:Collagen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003587:Cytomegalovirus / Q000235:genetics; D004355:Drug Stability; D015870:Gene Expression; D006358:Hot Temperature; D006801:Humans; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism* / Q000502:physiology*
D015335:Molecular Probes; D011994:Recombinant Proteins; D003094:Collagen