Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii

Autor: Stephen P. Mayfield, Miller Tran, Beth A. Rasala, Shengchun Guo, Ayswarya Ravi, Zivko L. Nikolov
Jazyk: angličtina
Rok vydání: 2018
Předmět:
0301 basic medicine
OPN
Chlamydomonas reinhardtii
law.invention
lcsh:Chemistry
law
ceramic hydroxyapatite
Osteopontin
lcsh:QH301-705.5
Spectroscopy
gallium
Chromatography
biology
Chemistry
phosphorylation
General Medicine
Recombinant Proteins
Computer Science Applications
Chloroplast
recombinant osteopontin
C. reinhardtii
protein
Ga-IMAC
purification
Biochemistry
Recombinant DNA
Phosphorylation
Biotechnology
inorganic chemicals
macromolecular substances
Article
Catalysis
Inorganic Chemistry
03 medical and health sciences
stomatognathic system
Genetics
Animals
Physical and Theoretical Chemistry
Molecular Biology
Binding selectivity
Chemical Physics
Organic Chemistry
RuBisCO
biology.organism_classification
030104 developmental biology
lcsh:Biology (General)
lcsh:QD1-999
Phosphoprotein
biology.protein
bacteria
Cattle
Other Biological Sciences
Other Chemical Sciences
Zdroj: International Journal of Molecular Sciences, Vol 19, Iss 2, p 585 (2018)
International journal of molecular sciences, vol 19, iss 2
International Journal of Molecular Sciences; Volume 19; Issue 2; Pages: 585
International Journal of Molecular Sciences
ISSN: 1422-0067
Popis: 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.
Databáze: OpenAIRE
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