Sieron A L, Fertala A, Ala-Kokko L, Prockop D J
Department of Biochemistry and Molecular Biology, Jefferson Medical College, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
J Biol Chem. 1993 Oct 5;268(28):21232-7.
A construct of the human gene for procollagen II (COL2A1) was prepared with an internal deletion of 5 kilobases that removed 12 exons coding for 291 amino acids from near the NH2 terminus of the triple helix. The construct was then used to transfect stably a human tumor cell line (HT-1080), and clones secreting internally deleted pro alpha 1(II) chain of procollagen II were isolated. The protein was purified, and the thermal stability of the triple-helical domain was assayed by brief protease digestion. The thermal stability of the internally deleted protein was the same as that of intact collagen II even though the triple helix was 39% shorter. Additionally, the thermal stability of the collagenase A fragment was the same as that of the collagenase A fragment of normal collagen II even though it was 38% shorter. Analysis of the results suggested that the thermal stabilities of large fragments of collagen II depended primarily on their contents of -Gly-Pro-Hyp-triplets corrected for length.
制备了人原胶原II（COL2A1）基因构建体，其内部缺失5千碱基，该缺失去除了三螺旋NH2末端附近编码291个氨基酸的12个外显子。然后使用该构建体稳定转染人肿瘤细胞系（HT-1080），并分离出分泌内部缺失的原胶原II的α1（II）链的克隆。纯化该蛋白质，并通过短暂的蛋白酶消化测定三螺旋结构域的热稳定性。尽管三螺旋短39%，但内部缺失蛋白质的热稳定性与完整胶原II相同。此外，胶原酶A片段的热稳定性与正常胶原II的胶原酶A片段相同，尽管它短38%。结果分析表明，胶原II大片段的热稳定性主要取决于其-Gly-Pro-Hyp-三联体的含量（校正长度后）。
