Research Description
Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products, such as proteins and viruses. Many nonionic surfactants (such as those belonging to the alkyl poly(ethylene oxide), CiEi family) and zwitterionic surfactants (such as dioctanoyl phosphatidyl choline, C8-lecithin) exhibit phase separation in water at suitable temperatures into a micelle-rich phase coexisting with a micelle-poor phase, thus forming two-phase aqueous micellar systems. The low surfactant concentrations and low phase separating temperatures, combined with the relatively mild and nondenaturing environment, suggests that these systems are potentially useful as extractant phases for the purification and concentration of proteins and other biomaterials. In particular, this type of liquid-liquid extraction, also known as cloud point extraction, should be particularly suitable for large-scale production due to its lower cost and easier scale-up, when compared to chromatographic operations.
Careful choices of the phase-forming surfactants or surfactant mixtures allow these two-phase aqueous micellar systems to separate biomolecules based on hydrophobicity, size, charge, or specific affinity. In one of the first applications envisioned for this type of systems, membrane-bound proteins were extracted preferentially to the micelle-rich phase, by virtue of the hydrophobic interactions between the oily micellar core and the hydrophobic patches on the surface of the protein. [C. Bordier, J. Biol. Chem., 256(4), 1604 (1981)]. Previous experimental and theoretical investigation of the partitioning behavior of several water-soluble proteins, as well as of several bacteriophages, showed that biomolecules tend to partition preferentially into the micelle-poor phase, based on their size, where they experience less excluded-volume interactions. [C.L. Liu, Y. Nikas, and D. Blankschtein, Biotechnol. Bioeng., 52, 185 (1996)]. Further improvement of the separation was achieved by introducing electrostatic interactions. By adding a small amount of the anionic surfactant sodium dodecylsulfate (SDS) to the nonionic surfactant system, the negatively-charged mixed micelles that form attract the positively-charged protein lysozyme, leading to an improved yield in the micelle-rich phase [D. Kamei, J. King, D. Wang, and D. Blankschtein, Biotechnol. Bioeng., 80, 233 (2002)]. Similarly, the negatively-charged enzyme glucose-6-phosphate dehydrogenase (G6PD) was shown to partition more extremely in two-phase aqueous micellar systems with added cationic cosurfactants, alkyltrimethylammonium bromide (CiTAB) [C. Yagui, H. Lam, D. Kamei, D. Wang, A. Pessoa-Jr, and D. Blankschtein, Biotechnol. Bioeng., 82, 445 (2003)].
In the present study, we attempted to improve the yield and specificity of the bioseparation by introducing specific affinity. In particular, we have investigated the affinity-enhanced partitioning of a model affinity-tagged protein - green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP) - in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl β-D-glucopyranoside (C10G1), which acts simultaneously as the phase-forming surfactant and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, in spite of the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a six-fold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp) as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein.
To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain. [H. Lam, M. Kavoosi, C. Haynes, D. Wang, and D. Blankschtein, Biotechnol. Bioeng., 89(4), 381 (2005)] More studies are underway to apply this promising system to a mixture derived from a real E. coli cell lysate, as well as to further improve and optimize the bioseparation.
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