Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.
Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Public Library of Science (PLoS)
2016-01-01
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| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC4883780?pdf=render |
| _version_ | 1818944491975868416 |
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| author | Minh Tan Nguyen Martin Krupa Bon-Kyung Koo Jung-A Song Thu Trang Thi Vu Bich Hang Do Anh Ngoc Nguyen Taewook Seo Jiwon Yoo Boram Jeong Jonghwa Jin Kyung Jin Lee Heung-Bum Oh Han Choe |
| author_facet | Minh Tan Nguyen Martin Krupa Bon-Kyung Koo Jung-A Song Thu Trang Thi Vu Bich Hang Do Anh Ngoc Nguyen Taewook Seo Jiwon Yoo Boram Jeong Jonghwa Jin Kyung Jin Lee Heung-Bum Oh Han Choe |
| author_sort | Minh Tan Nguyen |
| collection | DOAJ |
| description | Human vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli. |
| first_indexed | 2024-12-20T07:44:04Z |
| format | Article |
| id | doaj.art-1929551d9cec407983d1217bd8a4714f |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-20T07:44:04Z |
| publishDate | 2016-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-1929551d9cec407983d1217bd8a4714f2022-12-21T19:48:02ZengPublic Library of Science (PLoS)PLoS ONE1932-62032016-01-01115e015629610.1371/journal.pone.0156296Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag.Minh Tan NguyenMartin KrupaBon-Kyung KooJung-A SongThu Trang Thi VuBich Hang DoAnh Ngoc NguyenTaewook SeoJiwon YooBoram JeongJonghwa JinKyung Jin LeeHeung-Bum OhHan ChoeHuman vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis and plays a central role in the process of tumor growth and metastatic dissemination. Escherichia coli is one of the most common expression systems used for the production of recombinant proteins; however, expression of human VEGF in E. coli has proven difficult because the E. coli-expressed VEGF tends to be misfolded and forms inclusion bodies, resulting in poor solubility. In this study, we successfully produced semi-preparative amounts of soluble bioactive human VEGF165 (hVEGF). We created seven N-terminal fusion tag constructs with hexahistidine (His6), thioredoxin (Trx), glutathione S-transferase (GST), maltose-binding protein (MBP), N-utilization substance protein A (NusA), human protein disulfide isomerase (PDI), and the b'a' domain of PDI (PDIb'a'), and tested each construct for soluble overexpression in E. coli. We found that at 18°C, 92.8% of the MBP-tagged hVEGF to be soluble and that this tag significantly increased the protein's solubility. We successfully purified 0.8 mg of pure hVEGF per 500 mL cell culture. The purified hVEGF is stable after tag cleavage, contains very low levels of endotoxin, and is 97.6% pure. Using an Flk1+ mesodermal precursor cell (MPC) differentiation assay, we show that the purified hVEGF is not only bioactive but has similar bioactivity to hVEGF produced in mammalian cells. Previous reports on producing hVEGF in E. coli have all been based on refolding of the protein from inclusion bodies. To our knowledge, this is the first report on successfully expressing and purifying soluble hVEGF in E. coli.http://europepmc.org/articles/PMC4883780?pdf=render |
| spellingShingle | Minh Tan Nguyen Martin Krupa Bon-Kyung Koo Jung-A Song Thu Trang Thi Vu Bich Hang Do Anh Ngoc Nguyen Taewook Seo Jiwon Yoo Boram Jeong Jonghwa Jin Kyung Jin Lee Heung-Bum Oh Han Choe Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. PLoS ONE |
| title | Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. |
| title_full | Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. |
| title_fullStr | Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. |
| title_full_unstemmed | Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. |
| title_short | Prokaryotic Soluble Overexpression and Purification of Human VEGF165 by Fusion to a Maltose Binding Protein Tag. |
| title_sort | prokaryotic soluble overexpression and purification of human vegf165 by fusion to a maltose binding protein tag |
| url | http://europepmc.org/articles/PMC4883780?pdf=render |
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