Affinity-based Aqueous Two-phase Micellar Extraction And Facilitated Protein Refolding By Like-charged Magnetic Nanoparticles

In the production of recombinant proteins, purification and refolding are two common problems confronted by technicians. As a result of the advancing biotechnology, more and more proteins are heterogeneously expressed with high yield, and therefore universal and fast techniques for protein purification and refolding need to be developed. Herein, an affinity aqueous two-phase micellar system was developed to enhance the purification of His-rich proteins and magnetic particles with high charge density were synthesized to facilitate protein refolding.Aqueous two-phase micellar system(ATPMS), an alternative to chromatography, has been considered as a promising liquid–liquid extraction technique for biomolecule purification. However, its application is mainly limited to the extraction of hydrophobic proteins due to its low selectivity for hydrophilic ones. To improve the specificity of ATPMSs, a novel nickel-chelated surfactant(TX-Ni) was firstly fabricated by coupling Triton X-114(TX-114), a commercial nonionic surfactant, with iminodiacetic acid(IDA), and chelating nickel ions. Experimental results indicated that TX-Ni and TX-114 formed mixed micelles in the aqueous solution. However, the existence of TX-Ni was not favorable for micelle formation and inhibited the aggregation of micelles, attributed to the steric and electrostatic repulsion of TX-Ni. In addition, hydrophilic IDA moieties increased the hydration of micelles, thus improving the cloud point. The effect of inorganic salts on cloud point followed the Hofmeister series, and kosmotropic ions could be used to ensure extraction is possible to operate under a lower temperature. Secondly, the efficiency of TX-Ni as an affinity surfactant was demonstrated by the partition of 6×His tagged enhanced green fluorescence protein(EGFP) and lysozyme. In 4%(w/w) aqueous Triton solutions, when the molar fraction of TX-Ni increased from 0 to 0.1, the partition coefficient of 6×His-tagged EGFP showed a 20-fold increase(from 0.60 to 12.42). Besides, both the p H and salt concentration exhibited significant influence on the partitioning behavior of EGFP, implying that the affinity interaction played a dominant role. Finally, the ATPMS composed of TX-Ni and TX-114 was used to extract EGFP from cell lysate, and a recovery field of over 80% with a purity of 70% was achieved. This affinity-based ATPMS is thus considered promising for providing a versatile platform for the separation of histidine-rich proteins.It has been reported that polymer microspheres grafted with polyelectrolytes can significantly inhibit like-charged protein aggregation and enhance the protein refolding. It was found that the microspheres with smaller size and higher charge density performed better in facilitating protein refolding. Herein, polyelectrolyte-grafted magnetic nanoparticles were prepared with Fe3O4 as cores and polyelectrolytes as shells. Two monomers, [3-(Methacryloylamino) propyl] trimethylammonium chloride(MAPTAC) and sodium methacrylate(MAA), were used to synthesize the cationic or anionic polyelectrolytes, respectively, via atom transfer radical polymerization(ATRP). It was found that high charge density was achieved by the ATRP reaction. The cationic Fe3O4@PMAPTAC magnetic particle showed high BSA adsorption capacity and could enhance the refolding of lysozyme; meanwhile the anionic Fe3O4@PMAA magnetic particle exhibited high lysozyme adsorption capacity and could facilitated the refolding of EGFP. After refolding, the particles could be recycled easily by magnetic separation. It should be noted that, the polymer chains must be long enough to overcome the nonspecific adsorption between the particle surface and proteins.