Reagents and equipment
-Hydroxysuccinimidyl ester (NHS)-activated Sepharose 4 Fast Flow resin was purchased from GE Healthcare (Piscataway, NJ). Recombinant human H-ras was purchased from Calbiochem (San Diego, CA). [3
H]-farnesyl diphosphate ([3H]1-PP
) was purchased from American Radiolabeled Chemicals (St. Louis, MO). Farnesyl diphosphate (1-PP
], geranylgeranyl diphosphate (2-PP
], azido-10,11-dihydrofarnesyl diphosphate (3-PP
], propargyloxygeranyl diphosphate (4-PP
], yeast PFTase [30
], human PGGTase-I [31
], eGFP-CVIA [9
], CDC42 [8
], GST-CIIS [32
], and tris(benzyltriazolylmethyl)amine (TBTA) [33
] were prepared as previously described. Ultrapure water was generated by a Milli-Q water purification system (Millipore Inc., Billerica, MA, R
> 12 MΩ·cm−1
Preparation of β-CD-Sepharose column
was synthesized as previously reported [27
], by sequentially adding 1 equiv each of 1,1-carbonyldiimidazole (CDI) and ethylenediamine to β-CD in pyridine. Conversion was confirmed by 1
H NMR spectroscopy (300 MHz, Varian Unity) and by electrospray ionization (ESI) MS (reflectron ESI-BioTOF II, Bruker Daltonics, Billerica, MA).
To prepare Sepharose-NHS resin for conjugation with β-CD-NH2
, 10 mL of resin suspension (w/18 μmol NHS/mL) was drained and washed twice with 10 mL of cold 1.0 mM HCl and 10 mL of 15 mM HEPES buffer (pH 7.5), alternatively [9
]. The drained resin was then directly transferred with stirring into a vial containing 6.0 mL of β-CD-NH2
solution (8 mg/mL in 15 mM HEPES buffer, pH 7.5). After 4 h of stirring at room temperature, 5.0 mL of ethanolamine was added to block unreacted NHS functional groups on the resin. After incubating overnight, a 1.0 mL aliquot of the β-CD-Sepharose suspension was washed with 15 mM HEPES buffer (pH 7.5) and transferred into a Tricorn 10/20 column (GE Healthcare). The resulting β-CD-Sepharose column was connected to the Agilent 1100 HPLC instrument for gradient fast protein liquid chromatography (FPLC). The column was washed with 1.0 M NH4
Cl (adjusted to pH 7.0 with 1 M NaOH; solvent B) for 60 min at a flow rate of 0.3 mL/min, and then equilibrated to 80 mM NH4
Cl (adjusted to pH 7.0 with 1 M NaOH; solvent A) prior to use.
Mutagenesis, overexpression and purification of mCherry-CVIA
A pRSET-B plasmid containing His6-mCherry was the kind gift of Prof. Roger Tsien (University of California, San Diego). A CVIA tag sequence was inserted at the C-terminus using PCR-based insertion (QuikChange, Stratagene, La Jolla, CA), using the forward-sense insertion primer CATGGACGAGCTGTACAAGtgcgtcatcgcaTAAtggaattcgaagcttgatcc (encoding the end of mCherry, the CVIA tag, the stop codon, and the beginning of the plasmid cloning site), as well as its complement. Successful insertion of the CVIA tag sequence was confirmed by DNA sequencing. The resulting plasmid construct was transformed into BL21(DE3)PlysS supercompetent cells (Novagen, Madison, WI) under heat shock for 30 s at 42 °C. To overexpress the mCherry-CVIA, LB media (250 mL), supplemented with ampicillin (0.10 mg/mL), was inoculated with a single colony of cells containing the mCherry-CVIA plasmid and grown with shaking overnight at 37 °C. The culture (10 mL) was added to 500 mL flasks containing 250 mL of LB, ampicillin (0.10 mg/mL), and chloramphenicol (0.035 mg/mL). After shaking at 37 °C for 2 h, the OD600 of the cells had increased to 0.6, and induction was initiated by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM. The cells were grown with shaking at 37 °C for 5 h after induction. The cells were recovered by centrifugation, and kept frozen at −80 °C after removing supernatant. The next day, wet cells were suspended in lysis buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 10 mM imidazole). After the cells were incubated for 30 min with lysozyme (1.0 mg/mL) and benzonase (1.0 mg/mL), they were lysed by sonication on ice, using six rounds of alternating 30 s pulses and 30 s cooling periods. The lysis extract was centrifuged (10,000 × g) for 30 min at 4 °C, and the supernatant was loaded directly onto a column containing 5 mL of Ni-NTA resin (Qiagen) that had been equilibrated with 50 mL equilibration buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 20 mM imidazole). After loading the supernatant, the column was washed with equilibration buffer, and protein product was eluted with elution buffer (50 mM NaH2PO4, pH 8.0; 300 mM NaCl; 250 mM imidazole).
Enzymatic reactions were conducted in 32 mL volumes, containing 50 mM Tris-HCl adjusted to pH 7.0, 10 mM MgCl2, 10 μM ZnCl2, 5 mM dithiothreitol (DTT), and 2.5 μM protein substrate. This mixture was incubated for 1 h at 25 °C in order to reduce any disulfide-linked protein aggregates before performing the prenylation. Prenyl diphosphate and the appropriate prenyl transferase (either yeast PFTase or PGGTase-I) were then added to final concentrations of 10 nM (with activity 0.089 U/mg) and 10 μM (activity 0.022 U/mg), respectively. The enzymatic reaction incubated at 32 °C for 4 h. In the case of protein prepared only for FPLC analysis, the protein was purified by gel filtration (NAP-5). In the case of samples purified for subsequent MS, the product mixture was concentrated to 500 μL by centrifugal dialysis (Amicon Ultra-15, MWCO 10.0 kDa, Millipore), and was desalted by three rounds of adding 15 mL of 20 mM Tris-HCl (pH 7.5) to the dialysis tube and concentrating to 500 μL, prior to FPLC purification.
FPLC separation of prenylated proteins
FPLC was performed on an Agilent (Santa Clara, CA) 1100 instrument equipped with a diode-array UV/vis detector. The same FPLC method was applied to all protein samples. After sample injection, the mobile phase was maintained at 100% solvent A for 20 min, followed by a linear gradient from 100% A to 100 % B over 50 min, ending with an isocratic elution with 100% solvent B for 20 min. After the run was complete, the column was returned to 100% solvent A.
Prenylation of mCherry-CVIA with [3H]-farnesyl diphosphate
For radiolabeling experiments, 42 μL of 60 mM Tris-HCl, pH 7.0, 12 mM MgCl2, 12 μM ZnCl2, 18 mM dithiothreitol (DTT), and 3.0 μM mCherry-CVIA, was incubated for 1 h at 25 °C. Then, [3H]1-PP (16.7 μM, 1 mCi/mL) and yeast PFTase (activity 0.089 U/mg) were added to final concentrations of 1.0 μM and 10 nM, respectively, to bring the total volume to 50 μL. The reaction tube was incubated at 32 °C for 2 h. Most unreacted [3H]1-PP was removed by gel filtration (NAP-5, GE Healthcare). The entire 50 μL reaction volume was added to the column, and was eluted with 20 mM Tris-HCl, pH 7.5. The first 500 μL to elute from the column was discarded, and then the next 300 μL was collected. This solution was concentrated to 10 μL by centrifugal dialysis (Microcon YM-10, MWCO 10 kDa; Millipore), the dialysis cartridge was washed with 4 × 10 μL, and washes were recombined with the retentate (50 μL total). This sample was injected directly onto the FPLC column as described above. Fractions (1 mL) were collected from the column output, and 500 μL of each fraction was placed in a scintillation vial containing 5 mL of scintillation fluid. The radioactivity of the fractions was then measured using a Beckman LS 3801 scintillation counter.
Analytical mass spectrometry of prenylated proteins
Mass spectrometry of mCherry-CVIA and mCherry-CVIA-1 was performed on a reflectron ESI-micrOTOF-Q (Bruker Daltonics). Each protein was separately subjected to FPLC on β-CD-Sepharose. After collecting eluted protein, the samples were thoroughly washed and concentrated by repeated centrifugal dialysis (Amicon Ultra-15, MWCO 10.0 kDa) with H2O/MeOH (90/10 v/v, 4.0 mL × 7). The washed proteins were further dialyzed (using a Slide-A-Lyzer 2K MWCO cassette, Pierce, Rockford, IL) against H2O/MeOH/AcOH (89/10/1 v/v/v, 2 L × 2) to prepare them for ESI-MS injection. Protein was injected at a concentration of 20–30 μM into a flow of H2O/CH3CN/formic acid (64.9/35.0/0.1 v/v/v) at a flow rate of 0.1 mL/min. Ion series from m/z peaks were deconvoluted using Bruker DataAnalysis software.
Immobilization of mCherry-CVIA-azide onto alkyne-functionalized agarose beads
Alkyne-functionalized agarose beads were prepared according to a previously published protocol [9
]. To immobilize mCherry-CVIA-3
onto these beads, 30 μL of a 45 μM solution of mCherry-CVIA-3
in 20 mM Tris-HCl, pH 7.5, collected directly from purification on β-CD-Sepharose, was added to 100 μL of beads suspended in 50 mM phosphate buffer (pH 7.3). CuSO4
, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and TBTA were added to this mixture to final concentrations of 1.0 mM, 1.0 mM, and 100 μM, respectively. The reaction was allowed to proceed for 4 h at room temperature. As a negative control, non-prenylated mCherry-CVIA (lacking an azide group) was combined with alkyne-functionalized beads under the same reaction conditions. After the reaction, beads were washed with 1 M NaCl, 50 mM phosphate buffer (pH 7.3, 200 μL × 3). Fluorescence and bright-field images of these agarose beads were obtained with a Nikon Eclipse TE200 inverted microscope. Fluorescence images were obtained with a red filter set (excitation bandpass 540–590 nm, emission bandpass 600–650 nm).
β-CD-Sepharose FPLC purification of prenylated mCherry-CVIA from crude cell lysate
Pelleted HeLa cells (from 0.5 mL suspension culture, National Cell Culture Center) was resuspended in 2.5 mL of freeze-thaw buffer (10 mM Tris, pH 7.8; 0.6 M KCl; 2.8 M glycerol; 6.7 mM pefabloc; 40 μg/mL leupeptin; 40 mg/mL pepstatin; 20 μg/mL aprotinin). The cells were lysed by 5 alternating rounds of flash freezing and thawing in a 30°C water bath followed by brief vortexing. The protein concentration was determined by Bradford assay to be 2.7 mg/mL. Varying amounts of mCherry-CVIA-1 were then doped into aliquots of the crude cell lysate to final concentrations of 8.8%, 2.0%, and 0.4% of total protein concentration. These lysate samples were injected directly onto a β-CD-Sepharose column and analyzed by FPLC as described above. Fractions were collected, desalted and concentrated by centrifugal microdialysis (Microcon YM-10), and analyzed by 15% acrylamide, Tris-glycine SDS-PAGE. The gel was stained with Coomassie Brilliant Blue for imaging.