Purification of prenylated proteins by affinity chromatography on cyclodextrin-modified agarose

Jinhwa A. Chung, James W. Wollack, Marisa L. Hovlid, Ayse Okesli, Yan Chen, Joachim D. Mueller, Mark D. Distefano, T. Andrew Taton

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with β-cyclodextrin (β-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a β-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on β-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, β-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.

Original languageEnglish (US)
Pages (from-to)1-8
Number of pages8
JournalAnalytical Biochemistry
Volume386
Issue number1
DOIs
StatePublished - Mar 1 2009

Keywords

  • Affinity chromatography
  • Cyclodextrin
  • FPLC
  • Prenylation
  • Sepharose

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