Functional expression of Multidrug Resistance Protein 4 MRP4/ABCC4

Hardy, David, Bill, Roslyn M, Jawhari, Anass and Rothnie, Alice J (2019). Functional expression of Multidrug Resistance Protein 4 MRP4/ABCC4. SLAS Discovery ,

Abstract

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 over expression is imperative. Multidrug resistance protein 4 (MRP4) or ATP-binding cassette subfamily C member 4 (ABCC4) is a multi-transmembrane protein which 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 signalling 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 MRP4 expressed in Sf9 was functional using a fluorescent cAMP analogue (fluo-cAMP) instead of the traditional radiolabelled substrates. MRP4 transported fluo-cAMP in an ATP dependent manner. Specificity of functional expression of MRP4 was validated by the use of non-hydrolysable ATP analogues and MRP4 inhibitor MK571. Functionally expressed MRP4 in Sf9 cells can now be used in downstream processes such as solubilisation and purification in order to better understand its function and structure.

Publication DOI: https://doi.org/10.1177/2472555219867070
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences > Cellular and Molecular Biomedicine
Life & Health Sciences
Additional Information: © Sage 2019. The final publication is available via Sage at http://dx.doi.org/10.1177/2472555219867070
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Related URLs: https://journal ... cr_pub%3dpubmed (Publisher URL)
Accepted Date: 2019-07-08
Published Online Date: 2019-08-05
Authors: Hardy, David
Bill, Roslyn M ( 0000-0003-1331-0852)
Jawhari, Anass
Rothnie, Alice J ( 0000-0002-4259-7015)

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