Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin
Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from...
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2016-02-01
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| Series: | eLife |
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| Online Access: | https://elifesciences.org/articles/11621 |
| _version_ | 1828166509447348224 |
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| author | Emiko Mihara Hidenori Hirai Hideki Yamamoto Keiko Tamura-Kawakami Mami Matano Akira Kikuchi Toshiro Sato Junichi Takagi |
| author_facet | Emiko Mihara Hidenori Hirai Hideki Yamamoto Keiko Tamura-Kawakami Mami Matano Akira Kikuchi Toshiro Sato Junichi Takagi |
| author_sort | Emiko Mihara |
| collection | DOAJ |
| description | Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years. |
| first_indexed | 2024-04-12T02:03:55Z |
| format | Article |
| id | doaj.art-9b04a5fa9a914e4e98f0739e29aaa5bb |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-12T02:03:55Z |
| publishDate | 2016-02-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-9b04a5fa9a914e4e98f0739e29aaa5bb2022-12-22T03:52:37ZengeLife Sciences Publications LtdeLife2050-084X2016-02-01510.7554/eLife.11621Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albuminEmiko Mihara0Hidenori Hirai1Hideki Yamamoto2Keiko Tamura-Kawakami3Mami Matano4Akira Kikuchi5Toshiro Sato6Junichi Takagi7https://orcid.org/0000-0002-1219-475XLaboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita, JapanLaboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita, JapanDepartment of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, JapanLaboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita, JapanDepartment of Gastroenterology, Keio University School of Medicine, Tokyo, JapanDepartment of Molecular Biology and Biochemistry, Graduate School of Medicine, Osaka University, Suita, JapanDepartment of Gastroenterology, Keio University School of Medicine, Tokyo, JapanLaboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, Suita, JapanWnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years.https://elifesciences.org/articles/11621wntafaminserumpurificationepitope taglipid modification |
| spellingShingle | Emiko Mihara Hidenori Hirai Hideki Yamamoto Keiko Tamura-Kawakami Mami Matano Akira Kikuchi Toshiro Sato Junichi Takagi Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin eLife wnt afamin serum purification epitope tag lipid modification |
| title | Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin |
| title_full | Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin |
| title_fullStr | Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin |
| title_full_unstemmed | Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin |
| title_short | Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin |
| title_sort | active and water soluble form of lipidated wnt protein is maintained by a serum glycoprotein afamin α albumin |
| topic | wnt afamin serum purification epitope tag lipid modification |
| url | https://elifesciences.org/articles/11621 |
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