Microalgae in recent years have gained interest as a potential protein expression platform. Their ability to grow in simple media, undergo transformation into stable recombinant strains in short time and perform post-translational modifications while resisting viral or prion contamination give microalgae an edge over the bacterial and mammalian expression systems for selected protein products. Eukaryotic microalgae - Chlamydomonas reinhardtii, having a GRAS status, exemplifies the potential of this expression system to produce a variety of therapeutic proteins, including vaccines and monoclonal antibodies. Our study focuses on developing a downstream purification process for osteopontin (OPN), expressed in the chloroplast of C. reinhardtii. Widely present in the human body in bones, connective tissue and bodily fluids, osteopontin is a truly ubiquitous protein. Versatile in its functionality as an extracellular structural protein, cancer biomarker, promoter of cell-mediated immunity, osteopontin is a therapeutic brimming with potential in the healthcare industry. The chloroplast of C. reinhardtii has the ability to perform multiple phosphorylations crucial for the functional integrity of osteopontin. This study describes the development of a purification process leveraging the unique properties of the target recombinant protein. Osteopontin being a highly acidic protein, much like the majority of host protein in C. reinhardtii, combined with the susceptibility to proteolytic degradation makes the process development both challenging and intriguing. In an attempt to stabilize and partially purify osteopontin in the algal lysate, we screened several extract pretreatment methods. The impact of pretreatments on osteopontin yield, proteolytic stability and removal of host cell impurities will be discussed. We also compare and report capture chromatography of OPN on anion exchange, hydroxyapatite, and Fe- and Ga- immobilized metal affinity resins that were selected to leverage osteopontin’s unique biochemical and structural properties.

Developing a protein-specific purification approach for osteopontin expressed in chloroplast of Chlamydomonas reinhardtii

251st National Meeting (2016)

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