Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stabil...
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| Veröffentlicht in: | Applied microbiology and biotechnology 2013-08, Vol.97 (16), p.7253-7264 |
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| creator | Pan, Li Qiang Xie, Zhang Ming Tang, Xiao Jing Wu, Min Wang, Fu Rong Naranmandura, Hua Chen, Shu Qing |
| description | Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in
Escherichia coli
as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells. |
| doi_str_mv | 10.1007/s00253-012-4604-0 |
| format | Article |
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Escherichia coli
as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.</description><identifier>ISSN: 0175-7598</identifier><identifier>EISSN: 1432-0614</identifier><identifier>DOI: 10.1007/s00253-012-4604-0</identifier><identifier>PMID: 23208613</identifier><language>eng</language><publisher>Berlin/Heidelberg: Springer Berlin Heidelberg</publisher><subject>Analysis ; Antineoplastic Agents - isolation & purification ; Antineoplastic Agents - metabolism ; Antineoplastic Agents - pharmacology ; Apoptosis ; Biomedical and Life Sciences ; Biotechnologically Relevant Enzymes and Proteins ; Biotechnology ; Cancer ; Cell Line, Tumor ; Chemical bonds ; Chromatography ; Chromatography, Gel ; Collagen ; Collagen Type XVIII - genetics ; Collagen Type XVIII - isolation & purification ; Collagen Type XVIII - metabolism ; Collagen Type XVIII - pharmacology ; E coli ; Epithelial Cells - drug effects ; Epithelial Cells - physiology ; Escherichia coli ; Escherichia coli - genetics ; Gene Expression ; Genetic engineering ; Genetic recombination ; Humans ; Life Sciences ; Ligands ; Microbial Genetics and Genomics ; Microbiology ; Pharmaceutical industry ; Pharmaceuticals ; Protein Folding ; Proteins ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - isolation & purification ; Recombinant Fusion Proteins - metabolism ; Recombinant Fusion Proteins - pharmacology ; Studies ; TNF-Related Apoptosis-Inducing Ligand - genetics ; TNF-Related Apoptosis-Inducing Ligand - isolation & purification ; TNF-Related Apoptosis-Inducing Ligand - metabolism ; TNF-Related Apoptosis-Inducing Ligand - pharmacology ; Tumor necrosis factor ; Tumor necrosis factor-TNF ; Tumors</subject><ispartof>Applied microbiology and biotechnology, 2013-08, Vol.97 (16), p.7253-7264</ispartof><rights>Springer-Verlag Berlin Heidelberg 2012</rights><rights>COPYRIGHT 2013 Springer</rights><rights>Springer-Verlag 2013</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c543t-d7272360b2c035dab73d22e3c102d2252887567f75a5debe3d1a6cad6c01d2853</citedby><cites>FETCH-LOGICAL-c543t-d7272360b2c035dab73d22e3c102d2252887567f75a5debe3d1a6cad6c01d2853</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s00253-012-4604-0$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s00253-012-4604-0$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,780,784,27923,27924,41487,42556,51318</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23208613$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Pan, Li Qiang</creatorcontrib><creatorcontrib>Xie, Zhang Ming</creatorcontrib><creatorcontrib>Tang, Xiao Jing</creatorcontrib><creatorcontrib>Wu, Min</creatorcontrib><creatorcontrib>Wang, Fu Rong</creatorcontrib><creatorcontrib>Naranmandura, Hua</creatorcontrib><creatorcontrib>Chen, Shu Qing</creatorcontrib><title>Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1</title><title>Applied microbiology and biotechnology</title><addtitle>Appl Microbiol Biotechnol</addtitle><addtitle>Appl Microbiol Biotechnol</addtitle><description>Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in
Escherichia coli
as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.</description><subject>Analysis</subject><subject>Antineoplastic Agents - isolation & purification</subject><subject>Antineoplastic Agents - metabolism</subject><subject>Antineoplastic Agents - pharmacology</subject><subject>Apoptosis</subject><subject>Biomedical and Life Sciences</subject><subject>Biotechnologically Relevant Enzymes and Proteins</subject><subject>Biotechnology</subject><subject>Cancer</subject><subject>Cell Line, Tumor</subject><subject>Chemical bonds</subject><subject>Chromatography</subject><subject>Chromatography, Gel</subject><subject>Collagen</subject><subject>Collagen Type XVIII - genetics</subject><subject>Collagen Type XVIII - isolation & purification</subject><subject>Collagen Type XVIII - metabolism</subject><subject>Collagen Type XVIII - pharmacology</subject><subject>E coli</subject><subject>Epithelial Cells - drug effects</subject><subject>Epithelial Cells - physiology</subject><subject>Escherichia coli</subject><subject>Escherichia coli - genetics</subject><subject>Gene Expression</subject><subject>Genetic engineering</subject><subject>Genetic recombination</subject><subject>Humans</subject><subject>Life Sciences</subject><subject>Ligands</subject><subject>Microbial Genetics and Genomics</subject><subject>Microbiology</subject><subject>Pharmaceutical industry</subject><subject>Pharmaceuticals</subject><subject>Protein Folding</subject><subject>Proteins</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - isolation & purification</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Recombinant Fusion Proteins - pharmacology</subject><subject>Studies</subject><subject>TNF-Related Apoptosis-Inducing Ligand - genetics</subject><subject>TNF-Related Apoptosis-Inducing Ligand - isolation & purification</subject><subject>TNF-Related Apoptosis-Inducing Ligand - metabolism</subject><subject>TNF-Related Apoptosis-Inducing Ligand - pharmacology</subject><subject>Tumor necrosis factor</subject><subject>Tumor necrosis 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and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1</title><author>Pan, Li Qiang ; Xie, Zhang Ming ; Tang, Xiao Jing ; Wu, Min ; Wang, Fu Rong ; Naranmandura, Hua ; Chen, Shu Qing</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c543t-d7272360b2c035dab73d22e3c102d2252887567f75a5debe3d1a6cad6c01d2853</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>Analysis</topic><topic>Antineoplastic Agents - isolation & purification</topic><topic>Antineoplastic Agents - metabolism</topic><topic>Antineoplastic Agents - pharmacology</topic><topic>Apoptosis</topic><topic>Biomedical and Life Sciences</topic><topic>Biotechnologically Relevant Enzymes and Proteins</topic><topic>Biotechnology</topic><topic>Cancer</topic><topic>Cell Line, Tumor</topic><topic>Chemical bonds</topic><topic>Chromatography</topic><topic>Chromatography, Gel</topic><topic>Collagen</topic><topic>Collagen Type XVIII - genetics</topic><topic>Collagen Type XVIII - isolation & purification</topic><topic>Collagen Type XVIII - metabolism</topic><topic>Collagen Type XVIII - pharmacology</topic><topic>E coli</topic><topic>Epithelial Cells - drug effects</topic><topic>Epithelial Cells - physiology</topic><topic>Escherichia coli</topic><topic>Escherichia coli - genetics</topic><topic>Gene Expression</topic><topic>Genetic engineering</topic><topic>Genetic recombination</topic><topic>Humans</topic><topic>Life Sciences</topic><topic>Ligands</topic><topic>Microbial Genetics and Genomics</topic><topic>Microbiology</topic><topic>Pharmaceutical industry</topic><topic>Pharmaceuticals</topic><topic>Protein Folding</topic><topic>Proteins</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - isolation & purification</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Recombinant Fusion Proteins - pharmacology</topic><topic>Studies</topic><topic>TNF-Related Apoptosis-Inducing Ligand - genetics</topic><topic>TNF-Related Apoptosis-Inducing Ligand - isolation & purification</topic><topic>TNF-Related Apoptosis-Inducing Ligand - metabolism</topic><topic>TNF-Related Apoptosis-Inducing Ligand - pharmacology</topic><topic>Tumor necrosis factor</topic><topic>Tumor necrosis factor-TNF</topic><topic>Tumors</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Pan, Li Qiang</creatorcontrib><creatorcontrib>Xie, Zhang Ming</creatorcontrib><creatorcontrib>Tang, Xiao Jing</creatorcontrib><creatorcontrib>Wu, Min</creatorcontrib><creatorcontrib>Wang, Fu Rong</creatorcontrib><creatorcontrib>Naranmandura, Hua</creatorcontrib><creatorcontrib>Chen, Shu Qing</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE 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and biotechnology</jtitle><stitle>Appl Microbiol Biotechnol</stitle><addtitle>Appl Microbiol Biotechnol</addtitle><date>2013-08-01</date><risdate>2013</risdate><volume>97</volume><issue>16</issue><spage>7253</spage><epage>7264</epage><pages>7253-7264</pages><issn>0175-7598</issn><eissn>1432-0614</eissn><abstract>Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be a promising anticancer agent because its active form TRAIL trimer is able to induce apoptosis in different tumor cell lines while sparing normal cells. However, TRAIL trimer possesses a short half-life and low stability, which turns out to be a major obstacle for the development of clinical trials. In our present study, we constructed a recombined TRAIL trimer by genetic fusion of non-collagenous domain (NC1) of human collagen XVIII or its trimerization domain (TD) to C-terminus of TRAIL via a flexible linker, and then refolded the fusion proteins using a two-step refolding approach, namely a combination of dilution and gel filtration chromatography. As a result, both recombinant proteins, TRAIL-NC1 and TRAIL-TD, were expressed in
Escherichia coli
as inclusion bodies, and they exhibited difficultly to refold efficiently by conventional methods. Thereby, we applied a modified two-step refolding approach to refold fusion proteins. More than 55 % of TRAIL-NC1 and 90 % of TRAIL-TD protein activity was recovered during the two-step refolding approach, and their stability was also increased significantly. Also, size exclusion chromatography showed refolded TRAIL-NC1 was a trimer while TRAIL-TD, hexamer. However, both of them exerted good apoptosis activity on NCI-H460 cells.</abstract><cop>Berlin/Heidelberg</cop><pub>Springer Berlin Heidelberg</pub><pmid>23208613</pmid><doi>10.1007/s00253-012-4604-0</doi><tpages>12</tpages></addata></record> |
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| ispartof | Applied microbiology and biotechnology, 2013-08, Vol.97 (16), p.7253-7264 |
| issn | 0175-7598 1432-0614 |
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| subjects | Analysis Antineoplastic Agents - isolation & purification Antineoplastic Agents - metabolism Antineoplastic Agents - pharmacology Apoptosis Biomedical and Life Sciences Biotechnologically Relevant Enzymes and Proteins Biotechnology Cancer Cell Line, Tumor Chemical bonds Chromatography Chromatography, Gel Collagen Collagen Type XVIII - genetics Collagen Type XVIII - isolation & purification Collagen Type XVIII - metabolism Collagen Type XVIII - pharmacology E coli Epithelial Cells - drug effects Epithelial Cells - physiology Escherichia coli Escherichia coli - genetics Gene Expression Genetic engineering Genetic recombination Humans Life Sciences Ligands Microbial Genetics and Genomics Microbiology Pharmaceutical industry Pharmaceuticals Protein Folding Proteins Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - isolation & purification Recombinant Fusion Proteins - metabolism Recombinant Fusion Proteins - pharmacology Studies TNF-Related Apoptosis-Inducing Ligand - genetics TNF-Related Apoptosis-Inducing Ligand - isolation & purification TNF-Related Apoptosis-Inducing Ligand - metabolism TNF-Related Apoptosis-Inducing Ligand - pharmacology Tumor necrosis factor Tumor necrosis factor-TNF Tumors |
| title | Engineering and refolding of a novel trimeric fusion protein TRAIL-collagen XVIII NC1 |
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