Functional Expression of Multidrug Resistance Protein 4 MRP4/ABCC4

SLAS Discovery
David HardyAlice J Rothnie

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 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 flu...Continue Reading

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Citations

Nov 21, 2019·SLAS Discovery·Veli-Pekka Jaakola, Mariafrancesca Scalise
Feb 23, 2021·Molecular Biology Reports·Pérez-De Marcos Juan-CarlosTorres-Espíndola Luz-María

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Methods Mentioned

BETA
affinity purification
transfections
transfection
electrophoresis
Fluorescence
glycosylation

Software Mentioned

GraphPad Prism

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