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

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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.

Section snippets

Reagents and equipment

N-Hydroxysuccinimidyl ester (NHS)-activated Sepharose 4 Fast Flow resin was purchased from GE Healthcare (Piscataway, NJ, USA). Recombinant human H-ras was purchased from Calbiochem (San Diego, CA, USA). 3H-labeled farnesyl diphosphate ([3H]1-PP) was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Farnesyl diphosphate (1-PP) [29], geranylgeranyl diphosphate (2-PP) [29], azido-10,11-dihydrofarnesyl diphosphate (3-PP) [9], propargyloxygeranyl diphosphate (4-PP) [14], yeast

Preparation of β-CD-Sepharose for FPLC

The main objective of this study was to evaluate β-CD-modified Sepharose as an affinity medium for separating prenylated proteins from nonprenylated proteins and particularly for purifying the products of in vitro prenylation reactions from starting materials. In this study, CAAX-box-containing proteins were prenylated in vitro with either natural or nonnatural isoprenoid diphosphates, as shown in Fig. 1. These reactions routinely afford protein product contaminated with unreacted isoprenoid

Conclusions

Affinity chromatography of prenylated proteins on β-CD-Sepharose is a robust method for purifying both natural and engineered proteins from in vitro prenylation reactions. The method successfully isolates prenylated proteins from both nonprenylated starting materials and unreacted isoprenoid substrate. Although protein–matrix interactions play a role, separation in the system is driven by interactions between protein prenyl groups and column-bound cyclodextrin. Because in vitro prenylation is

Acknowledgments

T.A.T. and M.D.D. acknowledge support from the National Institutes of Health (CA122603 and GM058842). J.D.M. and Y.C. acknowledge support from the National Institutes of Health (GM64589) and the National Science Foundation (PHY-0346782). We thank Roger Tsien (University of California, San Diego) for providing the plasmid for mCherry, Stepan Lenevich for determining concentrations of prenyl diphosphates by NMR, and Dana Reed for helping with MS experiments.

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