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Plant J. 2009 Mar;57(5):932-44. doi: 10.1111/j.1365-313X.2008.03736.x. Epub 2008 Dec 4.

Purification of low-abundance Arabidopsis plasma-membrane protein complexes and identification of candidate components.

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1
Department of Plant Biology, Microbial and Plant Genomics Institute, University of Minnesota, St Paul, MN 55108, USA.

Abstract

Purification of low-abundance plasma-membrane (PM) protein complexes is a challenging task. We devised a tandem affinity purification tag termed the HPB tag, which contains the biotin carboxyl carrier protein domain (BCCD) of Arabidopsis 3-methylcrotonal CoA carboxylase. The BCCD is biotinylated in vivo, and the tagged protein can be captured by streptavidin beads. All five C-terminally tagged Arabidopsis proteins tested, including four PM proteins, were functional and biotinylated with high efficiency in Arabidopsis. Transgenic Arabidopsis plants expressing an HPB-tagged protein, RPS2::HPB, were used to develop a method to purify protein complexes containing the HPB-tagged protein. RPS2 is a membrane-associated disease resistance protein of low abundance. The purification method involves microsomal fractionation, chemical cross-linking, solubilization, and one-step affinity purification using magnetic streptavidin beads, followed by protein identification using LC-MS/MS. We identified RIN4, a known RPS2 interactor, as well as other potential components of the RPS2 complex(es). Thus, the HPB tag method is suitable for the purification of low-abundance PM protein complexes.

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