The preparation and development of cellular membrane affinity chromatography columns

Abstract

Cellular membrane affinity chromatography is a technique that is based on the immobilization of a target trans-membrane protein onto a stationary phase. The target protein is isolated by homogenization and solubilization of a source (e.g., cell line) followed by immobilization on either the immobilized artificial membrane-phosphatidyl choline (IAM-PC) stationary phase or the surface of an open tubular capillary during a dialysis step. The procedure typically takes 3–4 d for the IAM-PC stationary phase, whereas the open-tubular method takes an extra week for the preparation of the capillary. The resulting columns can then be used to characterize binding sites on the target protein through frontal chromatographic and/or nonlinear chromatographic studies using a wide variety of ligands including small molecules and polypeptides. The columns have been used in drug discovery as well as in the screening of tobacco smoke condensates.

Cellular membrane affinity chromatography (CMAC) in drug discovery from complex natural matrices

“…The process of preparing CMAC columns has been described in detail elsewhere by Moaddel and Wainer [11]. In this review, critical steps of column preparation are described and particular attention is paid to issues that have the most critical impact on the process of developing a functional CMAC.…”

“…Since the successful immobilization of nAChRs on the IAM stationary phase, multiple transmembrane proteins have been used to prepare CMACs with IAM.PC (immobilized artificial membrane phosphatidylcholine). The examples of transmembrane proteins immobilized on IAM.PC particles include: ligand-gated ion channels ( x  y nAChR x = 3,4,7; y = 2,3,4; GABA A , NMDA, combination of nAChR, NMDA) [11,13,[15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30], G-protein coupled receptors (opioid, -adrenergic, P2Y1, Histamine 1-4, CB 1 , CB 2 ) [31][32][33][34][35] and drug transporters (ABC: Pgp, MRP1, MRP2, BCRP; SLC: hOCT and hOCT SNPs, hOAT) [36][37][38][39]. IAM.PC mimics the cell membrane environment and keeps the experimental conditions close to the physiological state.…”

“…Introducing chemical changes in the immobilization process of enzymes may lead to irreversible modifications to protein structure and result in failure to develop functional assay [11]. It is important to keep boundary lipids surrounding the transmembrane protein intact as the subtle interactions between transmembrane domains and these lipids influences how the potential ligands bind to the protein.…”

“…It must be remembered that in the process of transmembrane protein immobilization, the cell membrane fragments and not the pure proteins are immobilized [7]. Stripping off the boundary lipids would not only lead to changing the receptor binding activity but also would probably result in the immobilization of protein aggregates, that would form during the immobilization process [11]. The aggregates would develop because nonpolar amino acid residues, forming the transmembrane domains would bury in the interior of the aggregates not to get exposed to an aqueous environment.…”

“…The aggregates would develop because nonpolar amino acid residues, forming the transmembrane domains would bury in the interior of the aggregates not to get exposed to an aqueous environment. The immobilization of cell membrane fragments with functional transmembrane receptors can be achieved in the process of cellular membrane affinity chromatography (CMAC) column preparation [11]. Successful immobilization of transmembrane fragments will result in obtaining an assay that enables the study of ligand-receptor interactions in close to physiological conditions (Figure 1).…”