Cloning and expression of human islet amyloid polypeptide in cultured cells
Cloning and expression of human islet amyloid polypeptide in cultured cells
复制标题
DOI：
10.1016/j.bbrc.2007.03.016
复制
发表时间：
2007-05-11
影响因子：
3.1
通讯作者：
Singh, Shashi
中科院分区：
文献类型：
--
作者：
Bhattacharya, Susinjan;Latha, J. Naveena Lavanya;Singh, Shashi
在细胞中克隆淀粉样蛋白的努力经常导致细胞死亡。我们成功地克隆和表达重组人胰岛淀粉样多肽（hIAPP）在培养的哺乳动物细胞。胰淀素被分泌，形成对靶细胞如大鼠和人的0细胞有毒的原纤维。该研究包括在荧光蛋白载体中克隆全长胰淀素，然后转染到哺乳动物细胞中。用重组人胰淀素转染的细胞分泌对应于37个氨基酸的天然成熟IAPP的翻译蛋白。将分泌出细胞的成熟IAPP纯化，并通过MALDI-TOF/TOF-MS和Western印迹进行表征。纯化的IAPP形成原纤维，如通过硫黄素-T荧光和AFM所见，并且这些原纤维对胰腺细胞系RIN 5 mf细胞具有细胞毒性。(c)2007年爱思唯尔公司All rights reserved.
Efforts to clone amyloidogenic proteins in the cells often have resulted in cell death. We report successful cloning and expression of recombinant human islet amyloid polypeptide (hIAPP) in cultured mammalian cells. Amylin gets secreted, forms fibrils that are toxic to target cells like 0 cells of rat and human. The study involves cloning of full-length amylin in fluorescent protein vector followed by transfection into mammalian cells. The transfected cells with recombinant human amylin, secrete the translated protein corresponding to 37-amino acid native mature IAPP. The mature IAPP secreted out of the cell is purified and characterized by MALDI-TOF/TOF-MS and Western blotting. Purified IAPP forms fibrils as seen by Thioflavin-T fluorescence and AFM, and these fibrils were cytotoxic towards pancreatic cell line RIN5mf cells. (c) 2007 Elsevier Inc. All rights reserved.