Functional expression of Multidrug Resistance Protein 4 MRP4/ABCC4

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 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.

Publication DOI: https://doi.org/10.1177/2472555219867070
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences
Additional Information: This article is distributed under the terms of the Creative Commons Attribution 4.0 License (http://www.creativecommons.org/licenses/by/4.0/) 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 (https://us.sagepub.com/en-us/nam/open-access-at-sage). 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 (RG110156).
Uncontrolled Keywords: ABC transporter,fluorescence,membrane protein expression,vesicular transport assay,Biotechnology,Analytical Chemistry,Biochemistry,Molecular Medicine
Publication ISSN: 2472-5552
Full Text Link:
Related URLs: https://journal ... cr_pub%3dpubmed (Publisher URL)
http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2019-12-01
Published Online Date: 2019-08-05
Accepted Date: 2019-07-08
Authors: Hardy, David
Bill, Roslyn M (ORCID Profile 0000-0003-1331-0852)
Jawhari, Anass
Rothnie, Alice J (ORCID Profile 0000-0002-4259-7015)

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