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 β cells of r...
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| Veröffentlicht in: | Biochemical and biophysical research communications 2007-05, Vol.356 (3), p.622-628 |
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| container_title | Biochemical and biophysical research communications |
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| creator | Bhattacharya, Susinjan Naveena Lavanya Latha, J. Kumresan, R. Singh, Shashi |
| description | 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 β 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. |
| doi_str_mv | 10.1016/j.bbrc.2007.03.016 |
| format | Article |
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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 β 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. 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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 β 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.</description><subject>AFM</subject><subject>Amyloid - biosynthesis</subject><subject>Amyloid - genetics</subject><subject>Amyloid fibrils</subject><subject>Amyloid toxicity</subject><subject>Amyloidogenesis</subject><subject>Animals</subject><subject>Apoptosis</subject><subject>Cell Survival</subject><subject>Cells, Cultured</subject><subject>Cercopithecus aethiops</subject><subject>CHO Cells</subject><subject>Cloning, Molecular</subject><subject>COS Cells</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>Escherichia coli - metabolism</subject><subject>Humans</subject><subject>Islet Amyloid Polypeptide</subject><subject>Microscopy, Atomic Force</subject><subject>Protein purification</subject><subject>Rats</subject><subject>Recombinant hIAPP</subject><subject>Spectrometry, Fluorescence</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</subject><subject>Thiazoles</subject><subject>Thioflavin-T binding</subject><subject>Transfection</subject><subject>Transgenic expression</subject><issn>0006-291X</issn><issn>1090-2104</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2007</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kEFv1DAQhS1ERbeFP8AB-cQtYSZOvLHEBa1aqKjUC0jcLMeegFdJHOwEsf--jnYlbpxGevO9p5nH2FuEEgHlh2PZddGWFcC-BFFm6QXbISgoKoT6JdsBgCwqhT-u2U1KRwDEWqpX7Br3Yl83ldyxr4chTH76yc3kOP2dI6Xkw8RDz3-to5m4TwMt3IynIXjH5zCcZpoX74j7idt1WNZIjlsahvSaXfVmSPTmMm_Z9_u7b4cvxePT54fDp8fCirZZil4o2_S1dKjAtRaxM6qupGjEvgVHjTG1yKsOW5RtDflWoZx0yhJltgVxy96fc-cYfq-UFj36tF1gJgpr0qhkTsINrM6gjSGlSL2eox9NPGkEvVWoj3qrUG8VahA6S9n07pK-diO5f5ZLZxn4eAYo__jHU9TJeposOR_JLtoF_7_8Z0K_gbo</recordid><startdate>20070511</startdate><enddate>20070511</enddate><creator>Bhattacharya, Susinjan</creator><creator>Naveena Lavanya Latha, J.</creator><creator>Kumresan, R.</creator><creator>Singh, Shashi</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QO</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope></search><sort><creationdate>20070511</creationdate><title>Cloning and expression of human islet amyloid polypeptide in cultured cells</title><author>Bhattacharya, Susinjan ; Naveena Lavanya Latha, J. ; Kumresan, R. ; Singh, Shashi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c385t-f39c5f46d190d8c11ba9426353780de5aa43190b181684017339d6d9cee11b803</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2007</creationdate><topic>AFM</topic><topic>Amyloid - biosynthesis</topic><topic>Amyloid - genetics</topic><topic>Amyloid fibrils</topic><topic>Amyloid toxicity</topic><topic>Amyloidogenesis</topic><topic>Animals</topic><topic>Apoptosis</topic><topic>Cell Survival</topic><topic>Cells, Cultured</topic><topic>Cercopithecus aethiops</topic><topic>CHO Cells</topic><topic>Cloning, Molecular</topic><topic>COS Cells</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>Escherichia coli - metabolism</topic><topic>Humans</topic><topic>Islet Amyloid Polypeptide</topic><topic>Microscopy, Atomic Force</topic><topic>Protein purification</topic><topic>Rats</topic><topic>Recombinant hIAPP</topic><topic>Spectrometry, Fluorescence</topic><topic>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization</topic><topic>Thiazoles</topic><topic>Thioflavin-T binding</topic><topic>Transfection</topic><topic>Transgenic expression</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bhattacharya, Susinjan</creatorcontrib><creatorcontrib>Naveena Lavanya Latha, J.</creatorcontrib><creatorcontrib>Kumresan, R.</creatorcontrib><creatorcontrib>Singh, Shashi</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><jtitle>Biochemical and biophysical research communications</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bhattacharya, Susinjan</au><au>Naveena Lavanya Latha, J.</au><au>Kumresan, R.</au><au>Singh, Shashi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cloning and expression of human islet amyloid polypeptide in cultured cells</atitle><jtitle>Biochemical and biophysical research communications</jtitle><addtitle>Biochem Biophys Res Commun</addtitle><date>2007-05-11</date><risdate>2007</risdate><volume>356</volume><issue>3</issue><spage>622</spage><epage>628</epage><pages>622-628</pages><issn>0006-291X</issn><eissn>1090-2104</eissn><abstract>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 β 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.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>17374526</pmid><doi>10.1016/j.bbrc.2007.03.016</doi><tpages>7</tpages></addata></record> |
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| ispartof | Biochemical and biophysical research communications, 2007-05, Vol.356 (3), p.622-628 |
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| subjects | AFM Amyloid - biosynthesis Amyloid - genetics Amyloid fibrils Amyloid toxicity Amyloidogenesis Animals Apoptosis Cell Survival Cells, Cultured Cercopithecus aethiops CHO Cells Cloning, Molecular COS Cells Cricetinae Cricetulus Escherichia coli - metabolism Humans Islet Amyloid Polypeptide Microscopy, Atomic Force Protein purification Rats Recombinant hIAPP Spectrometry, Fluorescence Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Thiazoles Thioflavin-T binding Transfection Transgenic expression |
| title | Cloning and expression of human islet amyloid polypeptide in cultured cells |
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