Separation Options for Phosphorylated Osteopontin from Transgenic Microalgae Chlamydomonas reinhardtii.

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.

Data on using single- and mixed-mode resins for capture chromatography of recombinant human thioredoxin from Escherichia coli.

This paper provides the data collected from screening chromatographic resins for their ability to bind and purify recombinant human thioredoxin from Escherichia coli lysate. This data was used by "Capture chromatography with mixed-mode resins: A case study with recombinant human thioredoxin from Escherichia coli" [1] to determine the optimal resin to use as a capture step to initiate downstream processing of thioredoxin. Five chromatography resins were screened using a 96-well filter plate to experiment on a wide range of pH and conductivity conditions in a shorter amount of time while saving on materials. Thioredoxin-producing E. coli was cultivated, harvested, and lysed according to Ravi et al [1]. Thioredoxin containing lysate was dialyzed into the binding conditions, pH from 5.0 to 9.0 and conductivity from 2.0 to 10.0 mS, applied to each resin and incubated with shaking for 0.5 h. Data gathered after the incubation period consisted of host cell protein and thioredoxin concentrations remaining in the supernatant, which was considered flowthrough for the remainder of this study. Samples containing high concentrations of thioredoxin after the experimental period indicate that thioredoxin did not bind to the resin at those conditions and should not be utilized as a capture step. Additionally, samples that contain low concentrations of host-cell proteins after the experimental period indicate large amounts of host-cell proteins bound to the resin. The corresponding conditions may not contribute to higher purity. Operating all screening experiments at small volumes allows for selecting optimal binding conditions while minimizing the burden on upfront biomass production.

Exploring the separation power of mixed-modal resins for purification of recombinant osteopontin from clarified Escherichia coli lysates.

Osteopontin (OPN) is a structural protein with potential value in therapeutic and diagnostic applications. Low titer, acidic isoelectric point, and the lack of well-defined secondary and tertiary structure were some of the challenges that complicated purification development of OPN from recombinant Escherichia coli lysates. Reported processes for OPN recovery from recombinant sources use nonorthogonal unit operations and often suffer from low yield. In this work, we expanded the search for an optimal OPN purification method by including mixed-modal resins with both ionic and hydrophobic properties (Capto adhere, HEA HyperCel, and PPA HyperCel). Plate-based high-throughput screening (HTS) platform revealed useful information about the interactions between the three different ligands and OPN as function of pH and ionic strength. The HTS data allowed the selection of OPN adsorption and elution conditions that were tested and optimized in a batch mode. In terms of purification factor and yield, HEA HyperCel performed significantly better than the other two mixed-modal resins. Pairing HEA HyperCel with a strong anion exchange step (Capto Q) resulted in a two-step purification process that achieved 45-fold purification of OPN with a final purity of 95% and 44% overall yield. The orthogonality provided by mixed-modal and ion exchange steps resulted in higher yield in fewer unit operations than reported processes. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2722, 2019.