Mono- and Diprenylated Rab7 GTPase
A R T I C L E S
mL) under an argon atmosphere. The mixture was left to stir for 1.25
h, and the solvent was removed by coevaporation with toluene (2 ×
40 mL). The compound was purified by flash chromatography on silica
gel. Beforehand, the silica gel was washed with dichloromethane/
methanol (30:1) containing 1% dimethylethylamine, followed by
dichloromethane/methanol (30:1), and finally pure dichloromethane.
For purification of the product, dichloromethane/methanol (15:1) was
used as eluent. The pure product was dissolved in dichloromethane
(30 mL) and washed with brine (20 mL). The aqueous layer was back-
extracted with dichloromethane (2 × 20 mL). The combined organic
layers were dried over Na2SO4 and filtered. The solvent was removed
under reduced pressure to yield 5.6 mg (77%) of the desired product
2.89 (m, 4H, ꢀ-CH2 Lys, â-CH2b Cys(GG), â-CH2b CysStBu), 2.80 (dd,
J ) 14.0, 7.6 Hz, 1H, â-CH2b Cys(GG)′), 1.95-2.13 (m, 24H, 12CH2
GG), 1.63-1.78 (m, 2H, â-CH2 Lys), 1.67-1.68 (4s, 12H, 4CH3), 1.60
(s, 18H, 6CH3), 1.39-1.48 (m, 4H, δ-, γ-CH2 Lys), 1.35 (s, 9H,
C(CH3)3). ESI-MS m/z: calcd for (M + H)+ C78H125N8O11S5 1509.8,
found 1510.0.
Cloning, Protein Expression, and Purification. REP-1 and
RabGGTase were purified as described previously.31,32 Briefly,
RabGGTase was purified from E. coli cells coexpressing R- and
â-subunits by a combination of metal-chelating and gel-filtration
chromatography.31 REP-1 was purified in a similar manner from yeast.32
Diprenylated wild-type Rab7:REP-1 complex was prepared by in vitro
prenylation as described.31
30b as a pale yellowish-green oil. Rf ) 0.38 (dichloromethane/methanol
1
(10:1)). [R]20 ) -37.6 (c ) 0.25, CH2Cl2/CH3OH (10:1)). H NMR
D
The coding region of the canine Rab7 gene truncated by six amino
acids was amplified by PCR using pET3a Rab7 plasmid as a template33
and the synthetic oligo-nucleotides 5′-ATTGGTACCCTTGGCAAAG-
CATGAGGTCTTGGCCCGGTCGTTC-3′ and T7 promoter as primers.
The PCR product was gel purified, digested with KpnI and NdeI, and
ligated into the pTYB1 (New England Biolabs) vector precut with the
same enzymes. The resulting plasmid (pTYB1-Rab7∆C6) was trans-
formed into E. coli BL21(DE3) cells, and transformants were selected
on ampicillin (50 mg/L) agar plates. A single colony was inoculated
into 5 mL of LB medium containing 125 mg/L Ampicillin, and the
culture was grown overnight at 37 °C. This preculture was used to
seed 2 L of fresh LB medium (containing 125 mg/L Ampicillin), and
the culture was incubated at 37 °C until the absorbance at 600 nm
(OD600) reached 0.5-0.7. IPTG was added to a final concentration of
0.5 mM, and overnight (or 10-12 h) induction was performed at 20
°C. Cells were harvested by centrifugation (5000g, 20 min, 4 °C) and
washed once in wash buffer (10 mM Na-Phosphate, pH 7.2, 0.1 M
NaCl). The bacterial pellet was resuspended in lysis buffer (25 mM
Na-Phosphate, pH 7.5, 0.5 M NaCl, 0.5 mM PMSF, 2 mM MgCl2
and 2 µM GDP), and cells were lysed by passing them twice through
a Microfluidizer (Microfluidics). A fresh portion of 0.5 mM PMSF
and Triton X-100 (1% final concentration) were added. The lysate was
cleared by ultracentrifugation (30 000g, 40min, 4 °C). An appropriate
amount of chitin beads, equilibrated with lysis buffer containing 1%
Triton X-100, was added to the supernatant, and the mixture was
incubated for 2 h on a rotating wheel at 4 °C. The beads were isolated
by centrifugation (2500g, 5 min, 4 °C) and washed 4 times with lysis
buffer containing 1% Triton X-100 followed by 4 times washing with
buffer without the detergent. Cleavage of the fusion protein was induced
by adding powdered MESNA to the beads suspension to a concentration
of 0.5 M and overnight incubation at room temperature. The supernatant
was collected by centrifugation and passed over a gel filtration column
equilibrated with ligation buffer (10 mM Na-Phosphate, pH 7.5, 0.1
mM MgCl2, 2 µM GDP). The pooled fractions were concentrated to at
least 10 mg/mL and shock frozen in multiple aliquots using liquid
nitrogen. The proteins could be stored at -80 °C for at least 2 years
without loss of ligation efficiency. Yields typically ranged from 10 to
30 mg of Rab7 protein thioester per liter of bacterial culture.
(500 MHz, CDCl3/CD3OD (10:1)): δ 8.54 (d, J ) 8.4 Hz, 1H, CH-2
dansyl), 8.32 (d, J ) 8.4 Hz, 1H, CH-8 dansyl), 8.19 (dd, J ) 7.2, 1.2
Hz, 1H, CH-4 dansyl), 7.58 (dd, J ) 8.4, 7.6 Hz, 1H, CH-7 dansyl),
7.54 (dd, J ) 8.4, 7.2 Hz, 1H, CH-3 dansyl), 7.23 (d, J ) 7.2 Hz, 1H,
CH-6 dansyl), 5.23 (t, J ) 7.4 Hz, 1H, CdCH-CH2-S GG), 5.07-
5.13 (m, 3H, 3CH GG), 4.78 (dd, J ) 7.4, 5.0 Hz, 1H, R-CH CysStBu),
4.59 (dd, J ) 8.0, 6.0 Hz, 1H, R-CH Cys(GG)), 4.51 (dd, J ) 5.2 Hz,
1H, R-CH Ser), 4.43 (dd, J ) 5.2 Hz, 1H, R-CH Ser′), 4.26 (dd, J )
8.2, 5.8 Hz, 1H, R-CH Lys), 3.87-3.93 (m, 2H, 2â-CH2a Ser), 3.77-
3.83 (m, 3H, 2â-CH2b Ser, R-CH CysStBu′), 3.75 (s, 3H, OCH3), 3.14-
3.26 (m, 4H, CH2-S GG, 2â-CH2a CysStBu), 3.08 (dd, J ) 13.8, 7.4
Hz, 1H, â-CH2b CysStBu), 2.99-3.04 (m, 1H, â-CH2a Cys(GG)), 2.90
(s, 6H, N(CH3)2), 2.84-2.90 (m, 4H, ꢀ-CH2 Lys, â-CH2b Cys(GG),
â-CH2b CysStBu′), 1.95-2.13 (m, 12H, 6CH2 GG), 1.71-1.83 (m, 2H,
â-CH2 Lys), 1.67 (s, 6H, 2CH3), 1.60 (s, 9H, 3CH3), 1.37-1.52 (m,
4H, δ-, γ-CH2 Lys), 1.35 (s, 9H, C(CH3)3), 1.32 (s, 9H, C(CH3)3). ESI-
MS m/z: calcd for (M + H)+, C62H101N8O11S6 1325.6; found, 1325.7.
S-tert-Butylthio-l-cysteyl-N(ꢀ)-5-(dimethylamino)-naphthalene-1-
sulfonyl-l-lysyl-l-seryl-S-geranylgeranyl-l-cysteyl-l-seryl-S-gera-
nylgeranyl-l-cysteine Methyl Ester (H-Cys(StBu)-Lys(dans)-Ser-
Cys(GG)-Ser-Cys(GG)-OMe) (30c). At room temperature, Fmoc-
Cys(StBu)-Lys(dansyl)-Ser-Cys(GG)-Ser-Cys(GG)-OMe 29c (10.0 mg,
5.8 µmol) was dissolved in dry dichloromethane/diethylamine (2:1; 1.0
mL) under an argon atmosphere. The mixture was left to stir for 1.25
h, and the solvent was removed by coevaporation with toluene (2 ×
40 mL). The compound was purified by flash chromatography on silica
gel. Beforehand, the silica gel was washed with dichloromethane/
methanol (30:1) containing 1% dimethylethylamine, followed by
dichloromethane/methanol (30:1), and finally pure dichloromethane.
For purification of the product, dichloromethane/methanol (15:1) was
used as eluent. The pure product was dissolved in dichloromethane
(30 mL) and washed with brine (20 mL). The aqueous layer was back-
extracted with dichloromethane (2 × 20 mL). The combined organic
layers were dried over Na2SO4 and filtered. The solvent was removed
under reduced pressure to yield 8.6 mg (99%) of the desired product
30c as a pale yellowish-green oil. Rf ) 0.35 (dichloromethane/methanol
1
(20:1)). [R]20 ) -31.4 (c ) 0.22, CH2Cl2/CH3OH (10:1)). H NMR
D
(400 MHz, CDCl3/CD3OD (10:1)): δ 8.53 (d, J ) 8.4 Hz, 1H, CH-2
dansyl), 8.31 (d, J ) 8.8 Hz, 1H, CH-8 dansyl), 8.19 (dd, J ) 7.2, 1.6
Hz, 1H, CH-4 dansyl), 7.57 (dd, J ) 8.8, 7.6 Hz, 1H, CH-7 dansyl),
7.53 (dd, J ) 8.4, 7.2 Hz, 1H, CH-3 dansyl), 7.21 (dd, J ) 7.6, 0.4
Hz, 1H, CH-6 dansyl), 5.22 (t, J ) 8.6 Hz, 1H, CdCH-CH2-S GG),
5.19 (t, J ) 9.0 Hz, 1H, CdCH-CH2-S GG′), 5.07-5.13 (m, 6H, 6CH
GG), 4.66 (dd, J ) 8.0, 5.2 Hz, 1H, R-CH Cys(GG)), 4.59 (dd, J )
8.0, 6.0 Hz, 1H, R-CH Cys(GG)′), 4.51 (dd, J ) 5.0 Hz, 1H, R-CH
Ser), 4.42 (dd, J ) 5.2 Hz, 1H, R-CH Ser′), 4.24 (dd, J ) 8.0, 6.0 Hz,
1H, R-CH Lys), 3.92 (dd, J ) 11.6, 3.6 Hz, 1H, â-CH2a Ser), 3.90 (dd,
J ) 11.2, 4.4 Hz, 1H, â-CH2a Ser′), 3.75-3.81 (m, 3H, 2â-CH2b Ser,
R-CH CysStBu), 3.74 (s, 3H, OCH3), 3.16-3.25 (m, 4H, CH2-SGG,
CH2a-S GG′, â-CH2a CysStBu), 3.11 (dd, J ) 13.4, 7.0 Hz, 1H, CH2b-S
GG), 3.01 (dd, J ) 13.8, 5.8 Hz, 1H, â-CH2a Cys(GG)), 2.94 (dd, J )
13.8, 5.4 Hz, 1H, â-CH2a Cys(GG)′), 2.89 (s, 6H, N(CH3)2), 2.83-
Detergent Screen. The collection of 72 detergents was from
Hampton detergent screen 1-3 (Hampton Research). A 40 µg (1.7
nmol) amount of Rab7∆6-MESNA thioester was mixed with 12 µg
(16 nmol) of peptide Cys-Lys(Dans)-Cys(GG)-OMe and the respective
detergent. The final detergent concentration was 3.8 times above its
respective critical micellar concentration. The mixture was incubated
at 30 °C for 10 h with gentle agitation. An aliquot of the mixture was
mixed with SDS-sample buffer and analyzed by SDS-PAGE.
(31) Kalinin, A.; Thoma, N. H.; Iakovenko, A.; Heinemann, I.; Rostkova, E.;
Constantinescu, A. T.; Alexandrov, K. Protein Exp. Purif. 2001, 22, 84-
91.
(32) Sidorovitch, V.; Niculae, A.; Kan, N.; Ceacareanu, A.; Alexandrov, K.
Protein Exp. Purif. 2002, 26, 50-58.
(33) Simon, I.; Zerial, M.; Goody, R. S. J. Biol. Chem. 1996, 271, 20470-
20478.
9
J. AM. CHEM. SOC. VOL. 126, NO. 50, 2004 16377