Protein partitioning in two-phase aqueous polymer systems

N. L. Abbott, D. Blankschtein, and T. A. Hatton, On protein partitioning in two-phase aqueous polymer systems, Bioseparation 1990, 1, 191-225. 

Experimental Observations on Protein Partitioning in Two-Phase Aqueous Polymer Systems... 

Experimental observations such as those presented in Figures 1 and 2 motivated the philosophy behind our theoretical formulation (21). In particular, the correlation observed between the overall sizes of the proteins and their partitioning behavior suggested that a coarse-grained view of these systems, rather than an atom-by-atom account, may be sufficient to describe the nature of the interactions responsible for the observed partitioning behaviors of these proteins. This realization prompted our decision to pursue (21,24) a scaling account (20,32-34) of protein partitioning in two-phase aqueous polymer systems. 

We have reviewed our recent theoretical and experimental studies of the physical mechanisms responsible for protein partitioning in two-phase aqueous polymer systems (21-25). It is gratifying to see that from a common physicd basis, our theoretical... 

Because the partitioning behavior of proteins in two-phase aqueous polymer systems reflects the relative interactions between the proteins and the two coexisting polymer solution phases, and because the independent control of the polymer concentration in only one of the two coexisting phases is not possible, we have explored an alternative experimental technique, namely, the measurement of the partitioning of proteins between an entangled PEO solution phase and an aqueous (polymer-free) phase using a diffusion cell (24). 

The formation of two aqueous phases can be exploited in the recovery of proteins using liquid-liquid extraction techniques. Many factors contribute to the distribution of a protein between the two phases. Smaller solutes, such as amino acids, partition almost equally between the two phases, whereas larger proteins are more unevenly distributed. This effect becomes more pronounced as protein size increases. Increasing the polymer molecular weight in one phase decreases partitioning of the protein to that phase. The variation in surface properties between different proteins can be exploited to improve selectivity and yield. The use of more hydrophobic polymer systems, such as fatty acid esters of PEG added to the PEG phase, favors the distribution of more hydrophobic proteins to this phase. In Fig. 10.13, partition coefficients for several proteins in a dextran-PEG system are given [27]. 

For the extraction of proteins, aqueous two-phase systems (ATPS) are preferred over organic solvents, which usually denature the proteins and render them biologically inactive. They consist of polyethylene glycol (PEG), and a salt (e.g., potassium phosphate) or dextran in water. At concentrations above a critical value, the mixture separates into two phases—one rich in PEG and the other in dextran or salt. In industrial systems, salts are more commonly used because they are relatively inexpensive as compared to dextran. The MW, charge and surface properties of the protein decide how the protein partitions in the system. The nature of the phase components, the MW of the polymer, and the concentration and type of salt used also affect the distribution. ... 

Separation by partition between two immiscible aqueous phases has been successfully applied to a large number of proteins, nucleic acids, and cellular particles such as mitochondria, chloroplasts, membrane vesicles, and whole cells. The phase systems are made up of two different water-soluble polymers, dextran and poly(ethylene glycol), dissolved in water and complemented with suitable buffers, salts, and sucrose. Highly selective adjustment of the distribution process is possible by linking an affinity... 

SIV Sivars, U. and Tjemeld, F., Mechanism of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins, Biochim. Biophys. Acta, 1474, 133, 2000. 

Aqueous two-phase systems have been used as a fast and effective process for separation of biomolecules (Gupta et al, 1999). The two-phase polymer systems are commonly formed by using two incompatible polymer/poly-mer or polymer/salt systems. Poly(ethylene) glycol-dextran systems are commonly used in two-phase separation. Partitioning is a complex process and depends on the surface properties of the proteins. Depending on its hydrophilic/hydrophobic properties the target product concentrates in one of the phases, while the impurities remain in another phase and can be easily removed. The recovery of the protein from the phase-forming polymer is the main bottleneck of the purification process. 

Pico, G., et al. Calorimetric investigation of the protein-flexible chain polymer interactions and its relationship with protein partition in aqueous two-phase systems. Int. J. Biol. Macromol. 40(3), 268-275 (2007)... 

The basis for the separation is that when two polymers, or a polymer and certain salts, are mixed together in water, they are incompatible, leading to the formation of two immiscible but predominantly aqueous phases, each rich in only one of the two components [Albertsson, op. cit. Kula, in Cooney and Humphrey (eds.), op. cit., pp. 451 71]. A phase diagram for a polyethylene glycol (PEG)-Dextran, two-phase system is shown in Fig. 22-85. Proteins are known to distribute unevenly between these phases. This uneven distribution can be used for the selective concentration and partial purification of the products. Partitioning between the two phases is controlled by the polymer molecular weight and concentration, protein net charge and... 

Jordan P, H Vilter (1991) Extraction of proteins from material rich in anionic mucilages partition and fractionation of vanadate-dependent bromoperoxidases from the brown algae Laminaria digitata and L. saccharina in aqueous polymer two-phase systems. Biochim Biophys Acta 1073 98-106. 

Aqueous two-phase cultivation of plant cells was found to be possible when the culture conditions were optimized. The selection of proper polymers and the optimization of their concentrations are needed not only for cell growth, but also for the desirable product partition. Promising examples of the use of aqueous two-phase systems for in situ extraction have been reported recently. However, more detailed and broader studies are necessary to utilize fully the potential of aqueous two-phase systems in plant cell culture. Finally, we believe that the development of fully integrated processes for plant cell cultures will improve the production efficiency of different kinds of secondary metabolites, enzymes, and recombinant proteins. 

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