Functional expression of Multidrug Resistance Protein 4 MRP4/ABCC4

David Hardy, Roslyn M Bill, Anass Jawhari*, Alice J Rothnie*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


To study the function and structure of membrane proteins, high quantities of pure and stable protein are needed. One of the first hurdles in accomplishing this is expression of the membrane protein at high levels and in a functional state. Membrane proteins are naturally expressed at low levels, so finding a suitable host for overexpression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein that is able to transport a range of organic anionic compounds (both endogenous and xenobiotic) out of the cell. This versatile transporter has been linked with extracellular signaling pathways and cellular protection, along with conferring drug resistance in cancers. Here we report the use of MRP4 as a case study to be expressed in three different expression systems: mammalian, insect, and yeast cells, to gain the highest yield possible. Interestingly, using the baculovirus expression system with Sf9 insect cells produced the highest protein yields. Vesicular transport assays were used to confirm that MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabeled substrates. MRP4 transported fluo-cAMP in an ATP-dependent manner. The specificity of functional expression of MRP4 was validated by the use of nonhydrolyzable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilization and purification in order to better understand its function and structure.

Original languageEnglish
Pages (from-to)1000-1008
Number of pages9
JournalSLAS Discovery
Issue number10
Early online date5 Aug 2019
Publication statusPublished - 1 Dec 2019

Bibliographical note

This article is distributed under the terms of the Creative Commons Attribution 4.0 License ( which permits any use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (
Funding: This work was funded by a Biotechnology and Biological Sciences
Research Council Industrial Case Studentship (BB/L015846/1).
A.J.R. was also the recipient of a Royal Society Research Grant


  • ABC transporter
  • fluorescence
  • membrane protein expression
  • vesicular transport assay


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