Passa al contenuto
MilliporeSigma

Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

International journal of molecular sciences (2018-02-22)
Ayswarya Ravi, Shengchun Guo, Beth Rasala, Miller Tran, Stephen Mayfield, Zivko L Nikolov
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.

MATERIALI
N° Catalogo
Marchio
Descrizione del prodotto

Sigma-Aldrich
Anticorpo monoclonale ANTI-FLAG® M2-perossidasi (HRP), clone M2, purified immunoglobulin, buffered aqueous glycerol solution
SKU
Taglio della confezione
Disponibilità
Prezzo
Quantità
Millipore
ANTI-FLAG® M1 Agarose Affinity Gel
SKU
Taglio della confezione
Disponibilità
Prezzo
Quantità
Sigma-Aldrich
Anticorpo monoclonale ANTI-FLAG® M2, clone M2, purified immunoglobulin, buffered aqueous glycerol solution
SKU
Taglio della confezione
Disponibilità
Prezzo
Quantità