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