Masamichi Kamihira 1 Introduction

Aqueous two-phase extraction has appreciable advantages in terms of ease of scaling-up and its applicability to systems containing solids such as cell and cell debris. However, because it is not always possible to achieve an extreme partitioning of a target molecule into either phase, the introduction of an affinity ligand to the system has been examined as a means of enhancing the specificity of partitioning. Covalent binding of a ligand to a phase-constructing polymer (1-4; see also Chapters 29-31), and the use of ligand-bound chromatographic supports (5-7; see also Chapter 33) or a pH responsive polymer (8; see also Chapter 34), that partitions to the upper phase have been employed as ligand-introduction procedures. As described in Chapter 34, the method using a pH responsive polymer can realize easy recovery of the target protein as a precipitate by changing the pH after specific partitioning into upper-phase, and the ligand is also easily recovered and reused after dissociation of the target protein.

The other difficulty in aqueous two-phase extraction is slow-phase separation. A typical aqueous two-phase system can achieve rapid equilibrium with minimum mixing because the surface tension between the phases is relatively low. In contrast, the phase separation is very slow, at least under static condition. Especially in the case of PEG-Dextran and PEG-modified starch systems, the solution is viscous. Therefore, the phase separation is accelarated by cen-trifugation. Nevertheless, simple and fast phase separation method is still desired. As an approach to this, Wikstrom and Flygare et al. reported that the introduction of magnetic particles that partition to one of the phases to various aqueous two-phase systems drastically shortened the phase separation time

From: Methods in Biotechnology, Vol. 11: Aqueous Two-Phase Systems: Methods and Protocols Edited by: R. Hatti-Kaul © Humana Press Inc., Totowa, NJ

Fig. 1. Flow diagram of separation procedures using magnetic particles as ligand carrier in aqueous two-phase system.

when a magnetic field was applied (9,10). The aqueous two-phase system was recognized as magnetic aqueous two-phase system. They have also developed the apparatus for magnetic aqueous two-phase systems. In their case, however, magnetic particles were neither modified nor immobilized with an affinity ligand and the particles partitioned to the bottom phase in PEG-based systems.

In this chapter, new magnetic aqueous two-phase system that we developed (11) will be described. An affinity ligand was immobilized to fine magnetic particles, which partition to the upper phase in aqueous two-phase systems. By introducing ligand-bound magnetic particles to aqueous two-phase systems, not only can selective partitioning and easy recovery be expected, but also the elimination of cell debris and a shortening of the phase-separation time by the application of a magnetic field. A flow dia gram of the separation procedures using ligand-bound magnetic particles in aqueous two-phase system is given in Fig. 1. As a model separation system, human IgG was immobilized to the magnetic particles and staphyloccocal protein A produced by recombinant Escherichia coli was purified with this method.

0 0

Post a comment