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Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii
Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855807/ https://www.ncbi.nlm.nih.gov/pubmed/29462927 http://dx.doi.org/10.3390/ijms19020585 |
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author | Ravi, Ayswarya Guo, Shengchun Rasala, Beth Tran, Miller Mayfield, Stephen Nikolov, Zivko L. |
author_facet | Ravi, Ayswarya Guo, Shengchun Rasala, Beth Tran, Miller Mayfield, Stephen Nikolov, Zivko L. |
author_sort | Ravi, Ayswarya |
collection | PubMed |
description | Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN. |
format | Online Article Text |
id | pubmed-5855807 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-58558072018-03-20 Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii Ravi, Ayswarya Guo, Shengchun Rasala, Beth Tran, Miller Mayfield, Stephen Nikolov, Zivko L. Int J Mol Sci Article Correct folding and post-translational modifications are vital for therapeutic proteins to elicit their biological functions. Osteopontin (OPN), a bone regenerative protein present in a range of mammalian cells, is an acidic phosphoprotein with multiple potential phosphorylation sites. In this study, the ability of unicellular microalgae, Chlamydomonas reinhardtii, to produce phosphorylated recombinant OPN in its chloroplast is investigated. This study further explores the impact of phosphorylation and expression from a “plant-like” algae on separation of OPN. Chromatography resins ceramic hydroxyapatite (CHT) and Gallium-immobilized metal affinity chromatography (Ga-IMAC) were assessed for their binding specificity to phosphoproteins. Non-phosphorylated recombinant OPN expressed in E. coli was used to compare the specificity of interaction of the resins to phosphorylated OPN. We observed that CHT binds OPN by multimodal interactions and was better able to distinguish phosphorylated proteins in the presence of 250 mM NaCl. Ga-IMAC interaction with OPN was not selective to phosphorylation, irrespective of salt, as the resin bound OPN from both algal and bacterial sources. Anion exchange chromatography proved an efficient capture method to partially separate major phosphorylated host cell protein impurities such as Rubisco from OPN. MDPI 2018-02-16 /pmc/articles/PMC5855807/ /pubmed/29462927 http://dx.doi.org/10.3390/ijms19020585 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Ravi, Ayswarya Guo, Shengchun Rasala, Beth Tran, Miller Mayfield, Stephen Nikolov, Zivko L. Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title | Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title_full | Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title_fullStr | Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title_full_unstemmed | Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title_short | Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii |
title_sort | separation options for phosphorylated osteopontin from transgenic microalgae chlamydomonas reinhardtii |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5855807/ https://www.ncbi.nlm.nih.gov/pubmed/29462927 http://dx.doi.org/10.3390/ijms19020585 |
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