Affinity membrane chromatography principle

Ligand Ligate Spacer FIGURE 6.2 Principle of affinity membrane chromatography. 

We have already observed in Figures 4.1.28(b) and (c) how polymeric chains anchored on the surfaces of pores of a membrane can bind metals, etc., present in the solution flowing through the membrane pores. This concept essentially applied the principle of membrane chromatography developed first with affinity adsorption of proteins to solve the problem of removal of metals from a solution (Brandt et al., 1988). AfBnity ligands attached to the membrane pore surfaces pick up proteins/antibodies/enzymes from the solution in a highly selective fashion. The membranes may be in the form of flat films (or hollow fibers, see Section 7.2). However, the amount of pore surface area available in one flat membrane (which is usually quite thin) is not substantial. Therefore a stack of membranes is used (Figure 7.1.27(b)). 

In principle any binding protein or receptor site or membrane fragment containing a receptor site can be purified by the use of an affinity medium containing the moiety with which the protein or receptor interacts. The extensive bibliography rapidly building up in this area of application indicates that, as with most other applications in affinity chromatography, the principle appears to be valid. 

The principles of affinity chromatography can be combined with other operations of purification to improve them (Labrou and Clonis 1994). Affinity partition combines the selectivity of affinity ligation with aqueous two-phase extraction (Kamihira et al. 1992 Kohler et al. 1991) and has been successfully employed in enzyme recovery and purification (Johansson and Tjerneld 1989 Schustolla et al. 1992) obtaining impressive increases in the partition coefficient (Eq. 2.16) and therefore in yield of enzyme recovery (Eq. 2.17). Affinity partition has also been combined with membrane separation (affinity ultrafiltration), where a soluble... 

The very same principle was applied by Suzuki et al. [19] for the purification of the placental insulin receptor, starting with a prepurified sample, obtained from a plasma membrane fraction of human placenta by a fractionation protocol including affinity chromatography on Sepharose-concanavalin A as the last step. The following purification step was carried out, using insulin bound to dextran (M 40000) as the biospecific, water-soluble carrier. After filtration of the dextran-insulin-receptor complex on an adequate gel and then dissociation by mild acidification, the insulin receptor was obtained in a purified form. This method permitted a 8700 fold purification, from crude plasma membrane and, even more remarkable, a 60-fold purification after the affinity chromatography step. 

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