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

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 beta-cyclodextrin (beta-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 beta-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 beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-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.

Fig. 1

Prenylation reactions purified in this study. PFTase and PGGTase-I accept their natural substrates (1-PP and 2-PP, respectively), but also catalyze the addition of isoprenoids containing non-natural functional groups.

Fig. 2

Preparation and use of β-CD-Sepharose for purification of prenylated proteins. In aqueous buffer, prenylated products are captured by the β-CD-modified support, but non-prenylated proteins bind poorly.

Fig. 3

FPLC analysis, using a β-CD-Sepharose column, of in vitro farnesylation reactions of between mCherry-CVIA and 1-PP, catalyzed by PFTase. (A) Pure mCherry-CVIA, prior to farnesylation. (B) Product mixture (containing both mCherry-CVIA and mCherry-CVIA-1) after prenylation. Based on peak integrations, 95% of mCherry-CVIA was prenylated under these conditions. (C) Fraction collected over t = 6–15 min from run B, reinjected onto the FPLC column. (D) Fraction collected over t = 42–60 min from run B, reinjected onto the FPLC column. Solid lines: Relative optical absorbance of eluent at λ = 586 nm, measured on-line. Dashed lines: Illustration of the NH₄Cl salt gradient.

Fig. 4

ESI-MS mass spectra of (A) mCherry-CVIA and (B) mCherry-CVIA-1, labeled with deconvoluted masses for each of the three peak series. In addition to an ion series corresponding to parent protein [M+H], the spectra also show ion series [M+H+98] and [M+H+2(98)]. This pattern has been previously observed and ascribed to associated H₂PO₄- ions [–47].

Fig. 5

FPLC analysis, using a β-CD-Sepharose column, of (solid line) farnesylation of mCherry-CVIA with PFTase and [³H]1-PP, and (dashed line) [³H]1-PP alone.

Fig. 6

Conjugation reactions of alkyne-modified agarose beads with (top) azide-functionalized mCherry-CVIA-3, and (bottom) non-functionalized mCherry-CVIA. Fluorescence images were acquired using identical illumination and imaging settings.

Fig. 7

β-CD-Sepharose FPLC of crude Hela cell lysate, spiked with different fractions of mCherry-CVIA-1 (relative to the total amount of protein in the lysate): (A) 8.8%; (B) 2.0%; (C) 0.4%.

Fig. 8

SDS PAGE analysis of mCherry-CVIA-1, purified from HeLa cell lysate by β-CD-Sepharose FPLC. Lane 1: pure mCherry-CVIA-1 (1 μg), prior to FPLC; lane 2: 8.8% mCherry-CVIA-1 in HeLa cell lysate (27 μg total); lane 3: fraction collected from FPLC, elution time 37–50 min.