Multiple protein stationary phases: a review

Abstract

Cellular membrane affinity chromatography stationary phases have been extensively used to characterize immobilized proteins and provide a direct measurement of multiple binding sites, including orthosteric and allosteric sites. This review will address the utilization of immobilized cellular and tissue fragments to characterize multiple transmembrane proteins co-immobilized onto a stationary phase. This approach will be illustrated by demonstrating that multiple transmembrane proteins were immobilized from cell lines and tissue fragments. In addition, the immobilization of individual compartments/organelles within a cell will be discussed and the changes in the proteins binding/kinetics based on their location.

Keywords: ATP-binding cassette transporters; Bioaffinity chromatography; G-protein coupled receptors; Ligand-gated ion channels.

Figure 1

The chromatographic traces obtained for 60 pM [3H]-EB on the CMAC(1321N1) column (panel A) and CMAC(A172) column (panel B) where A is the trace obtained using a mobile phase composed of ammonium acetate [10 mM, pH 7.4]; B is the trace obtained after the addition of 5 nM R-BTx to the mobile phase; and C is the trace obtained after the addition of 1 nM κ-BTx to the mobile phase. Reprinted with permission from [12].

Figure 2

Single frontal displacement studies of 8 compounds (4.25 μM), including etoposide, for the BCRP carried out on both CMAC(LN-229)-OT and NMAC(LN-229)-OT using ammonium acetate [10 mM, pH 7.4] in the presence of 2 μM verapamil and 0.75 μM etoposide as the mobile phase. The data was normalized to the change in breakthrough volume observed with etoposide and the relative changes from etoposide (0%) are reported. Reprinted with permission from [10].