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Int. J. Mol. Sci. 2018, 19(2), 585; https://doi.org/10.3390/ijms19020585

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

1
Department of Biological and Agricultural Engineering, Texas A&M University, College Station, TX 77843, USA
2
Triton Algae Innovations, San Diego, CA 92121, USA
3
California Center of Algae Biotechnology, University of California San Diego, San Diego, CA 92093, USA
*
Author to whom correspondence should be addressed.
Received: 21 November 2017 / Revised: 10 February 2018 / Accepted: 13 February 2018 / Published: 16 February 2018
(This article belongs to the Special Issue Recombinant Proteins)
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Abstract

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. View Full-Text
Keywords: recombinant osteopontin; C. reinhardtii; protein; phosphorylation; ceramic hydroxyapatite; gallium; Ga-IMAC; purification; OPN recombinant osteopontin; C. reinhardtii; protein; phosphorylation; ceramic hydroxyapatite; gallium; Ga-IMAC; purification; OPN
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Ravi, A.; Guo, S.; Rasala, B.; Tran, M.; Mayfield, S.; Nikolov, Z.L. Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii. Int. J. Mol. Sci. 2018, 19, 585.

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