Reconstitution and Transport Analysis of Eukaryotic Transporters in the Post-Genomic Era

Abstract

Measuring transport activity through reconstituted proteoliposomes is a key technique to resolve numerous problems found in the traditional methods. The system includes overexpression, purification, and reconstitution of transporters. Mixing of purified transporter with lipid and dilution below the critical micelle concentration result in rapid generation of proteoliposomes. Incubation of proteoliposomes in the presence of a driving force initiates substrate uptake. After starting the reaction, samples are passed through a gel filtration column to separate proteoliposomes from the reaction mixture. Here, we describe step-by-step procedures for such reconstitution assays.

Identification of Food-Derived Isoflavone Sulfates as Inhibition Markers for Intestinal Breast Cancer Resistance Proteins

“…Transport studies were performed using the membrane vesicles described above, as previously described (Omote and Moriyama, 2018). Briefly, 15 µL of reaction buffer (250 mM sucrose, 10 mM Tris-HCl, 10 mM MgCl 2 , 10 mM creatinine phosphate, and 100 µg/mL creatinine kinase, pH 7.4, in the presence of 4 mM ATP or AMP) containing test compounds (3 µM of DS and ES, 10 µM LY, and 5 µM N-methyl-quinidine [NMQ]), were preincubated for 5 min at 37 o C. To initiate transport, the reaction buffer was mixed with a vesicle suspension (5 µg protein) containing various concentrations of lapatinib, febuxostat, and KO143.…”