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Cloning, expression and purification of functionally active human angiopoietin-like protein 2
Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-li...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Springer International Publishing
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112196/ https://www.ncbi.nlm.nih.gov/pubmed/25077060 http://dx.doi.org/10.1186/2193-1801-3-337 |
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author | Farhat, Nada Mamarbachi, Aida M Thorin, Eric Allen, Bruce G |
author_facet | Farhat, Nada Mamarbachi, Aida M Thorin, Eric Allen, Bruce G |
author_sort | Farhat, Nada |
collection | PubMed |
description | Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-like protein 2 have been hindered by the lack of a source for the mature form of this protein. We describe a system that permits purification of the glycosylated form of human Angptl2 from conditioned media of stably transfected HEK 293 cells. To facilitate purification while retaining the integrity of Angptl2’s endogenous N-terminal secretion signal peptide, GST was fused downstream of the Angptl2 coding sequence. Secreted Angptl2-GST was purified using a one-step glutathione-affinity purification scheme. The purity and identity of the resulting protein were confirmed by SDS-PAGE, immunoblotting, and mass spectrometry. N-Glycosidase treatment reduced the apparent molecular mass of Angptl2-GST on SDS-PAGE, confirming its glycosylation state. Purified human Angptl2-GST stimulated both HUVEC migration and microtubule formation in vitro. The yield of Angptl2-GST obtained was in quantities suitable for multiple applications including functional in vitro and in vivo assays. |
format | Online Article Text |
id | pubmed-4112196 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Springer International Publishing |
record_format | MEDLINE/PubMed |
spelling | pubmed-41121962014-07-30 Cloning, expression and purification of functionally active human angiopoietin-like protein 2 Farhat, Nada Mamarbachi, Aida M Thorin, Eric Allen, Bruce G Springerplus Methodology Angiopoietin-like protein 2 (Angptl2) is a secreted glycoprotein that has been implicated in angiogenesis, inflammation and atherosclerosis as well as enhancing the survival of human hematopoietic stem cells. Glycosylation of Angptl2 is required for biological activity and studies of angiopoietin-like protein 2 have been hindered by the lack of a source for the mature form of this protein. We describe a system that permits purification of the glycosylated form of human Angptl2 from conditioned media of stably transfected HEK 293 cells. To facilitate purification while retaining the integrity of Angptl2’s endogenous N-terminal secretion signal peptide, GST was fused downstream of the Angptl2 coding sequence. Secreted Angptl2-GST was purified using a one-step glutathione-affinity purification scheme. The purity and identity of the resulting protein were confirmed by SDS-PAGE, immunoblotting, and mass spectrometry. N-Glycosidase treatment reduced the apparent molecular mass of Angptl2-GST on SDS-PAGE, confirming its glycosylation state. Purified human Angptl2-GST stimulated both HUVEC migration and microtubule formation in vitro. The yield of Angptl2-GST obtained was in quantities suitable for multiple applications including functional in vitro and in vivo assays. Springer International Publishing 2014-07-03 /pmc/articles/PMC4112196/ /pubmed/25077060 http://dx.doi.org/10.1186/2193-1801-3-337 Text en © Farhat et al.; licensee Springer. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. |
spellingShingle | Methodology Farhat, Nada Mamarbachi, Aida M Thorin, Eric Allen, Bruce G Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title | Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title_full | Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title_fullStr | Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title_full_unstemmed | Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title_short | Cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
title_sort | cloning, expression and purification of functionally active human angiopoietin-like protein 2 |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112196/ https://www.ncbi.nlm.nih.gov/pubmed/25077060 http://dx.doi.org/10.1186/2193-1801-3-337 |
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