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Updated: Sep 28, 2026

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein
10:43

Expression, Detergent Solubilization, and Purification of a Membrane Transporter, the MexB Multidrug Resistance Protein

Published on: December 3, 2010

Reconstitution of transport-active multidrug resistance protein 2 (MRP2; ABCC2) in proteoliposomes

Wolfgang Hagmann1, Jana Schubert, Jörg König

  • 1Abteilung Tumorbiochemie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.

Biological Chemistry
|September 12, 2002
PubMed

Insights

Researchers purified and reconstituted the apical multidrug resistance protein MRP2 (ABCC2) into proteoliposomes, demonstrating its active transport function for substrates like LTC4. This breakthrough enables further study of MRP2 interactions in polarized cells.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • The apical multidrug resistance protein MRP2 (ABCC2) functions as an ATP-dependent export pump for anionic conjugates in polarized cells.
  • MRP2 shares limited amino acid identity (48%) with its paralog, MRP1 (ABCC1).

Purpose of the Study:

  • To reconstitute purified recombinant MRP2 (ABCC2) into proteoliposomes to demonstrate its functional activity in substrate transport.
  • To characterize the kinetic parameters and inhibition of MRP2 transport.
  • To investigate the interaction of MRP2 with the chaperone calnexin.

Main Methods:

  • Purification of recombinant MRP2 (ABCC2).
  • Reconstitution of purified MRP2 into proteoliposomes.
  • Assay of substrate transport (LTC4) and ATP dependence.
  • Determination of kinetic parameters (Km) and inhibition by MK571.
  • Binding and co-reconstitution studies with calnexin.

Main Results:

  • Purified recombinant MRP2 reconstituted in proteoliposomes exhibited active substrate transport.
  • Kinetic analysis revealed Km values for ATP (560 µM) and LTC4 (450 nM).
  • Transport activity was dependent on MRP2 protein amount and inhibited by MK571.
  • MRP2 preferentially associates with calnexin, but calnexin is not essential for MRP2's LTC4 transport function.

Conclusions:

  • This study reports the first successful reconstitution of transport-active purified recombinant MRP2.
  • The characterized transport activity and inhibition provide a basis for further investigation.
  • Purified MRP2 can be used to identify additional interacting proteins in the apical membrane of polarized cells.

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