Cloning and expression of human islet amyloid polypeptide in cultured cells.
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 beta 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.
Bhattacharya
Susinjan
S
Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
Latha
J Naveena Lavanya
JN
Kumresan
R
R
Singh
Shashi
S
D000682:Amyloid / Q000096:biosynthesis / Q000235:genetics*; D000818:Animals; D052160:Benzothiazoles; D016466:CHO Cells; D019556:COS Cells; D002470:Cell Survival; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D058228:Islet Amyloid Polypeptide; D018625:Microscopy, Atomic Force; D051381:Rats; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013844:Thiazoles; D014162:Transfection
D000682:Amyloid; D052160:Benzothiazoles; D058228:Islet Amyloid Polypeptide; D013844:Thiazoles; C009462:thioflavin T