Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

Ayswarya Ravi,Stephen Mayfield,Miller Tran,Zivko Nikolov,Beth Rasala,Shengchun Guo

doi:10.3390/ijms19020585

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.

Highlights

The work horse of complex protein production for nearly 56% of recombinant proteins is the mammalian Chinese hamster ovary (CHO) cell line [1]
The transgenic cells of C. reinhardtii and E. coli expressing OPN were created by Mayfield’s group at California Center of Algae Biotechnology, University of California San Diego (UCSD) [24] and biomass was grown at Texas A&M University
C. reinhardtii OPN expression levels varied between biomass batches and was on average 4× lower than OPN in E. coli cells, as indicated by similar band densities of 4× and 16× diluted C. reinhardtii and E. coli cell lysates, respectively (Figure 1, lanes 2 & 3)

Summary

Introduction

The work horse of complex protein production for nearly 56% of recombinant proteins is the mammalian Chinese hamster ovary (CHO) cell line [1]. The ability of mammalian cells to properly fold and perform post-translational modifications (PTMs) of proteins, out-weighs the high cultivation costs and potential viral contamination issues. The chloroplast of C. reinhardtii has been utilized for the production of difficult-to-express proteins that require specific folding and PTMs to exhibit biological function [3,4,5,6,7]. One such mammalian protein is osteopontin (OPN), called bone sialoprotein-1 (BSP1), which is present in milk, bodily fluids and is responsible for bone development and regeneration. To take advantage of the diverse beneficial activities of OPN, a potential recombinant protein production system should be able to deliver optimal in vivo OPN phosphorylation

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