1284 J. Am. Chem. Soc., Vol. 122, No. 7, 2000
Van Hest et al.
mmol) was added. After 3 h of stirring, the mixture was poured into
aliquots of the resulting culture were prepared, and were supplemented
with 200 µL of 1 mg/mL (0.27 mM) L-methionine (1) (positive control),
DL-2-amino-5-hexenoic acid (2) (0.31 mM), DL-2-amino-5-hexynoic
acid (3) (0.31 mM), cis- or trans-DL-2-amino-4-hexenoic acid (4 or 5)
(0.31 mM), DL-6,6,6-trifluoro-2-amino hexanoic acid (6) (0.22 mM),
DL-2-aminoheptanoic acid (7) (0.28 mM), L-norvaline (8) (0.34 mM),
or L-norleucine (9) (0.31 mM), respectively. A culture lacking me-
thionine (or any analogue) served as the negative control. Protein
expression was induced by addition of isopropyl-â-D-thiogalactopyra-
noside (IPTG) to a final concentration of 0.4 mM. Samples were taken
every hour for 4 h, the OD600 was measured, and the samples were
sedimented. After the supernatant was decanted, the cell pellets were
3
0 mL of an ice/concentrated HCl 4/1 v/v solution, extracted with 60
mL of diethyl ether and dried overnight in the freezer over MgSO
The mixture was filtered, and the ether was evaporated to yield 7.22 g
4
.
1
(
76%) of 3-buten-1-ol 4-methylbenzenesulfonate as a yellow oil. H
NMR (CDCl ): δ 2.39-2.53 (m, 2H, J ) 6.5 and 6.9 Hz, CH sCHd
CH ; and s, 3H, CH -Ar), 4.08 (t, J ) 6.5 Hz, 2H, CH OSO ), 5.09-
.15 (m, 2H, J ) 10.4, J ) 16.6, J gem ) 3.1 Hz, CH sCHdCH ),
.57-5.82 (m, 1H, J ) 16.6, J ) 6.9 Hz, CH sCHd
3
2
2
3
2
2
5
5
Z
E
2
2
Z
) 10.4, J
E
2
CH ), 7.38 and 7.72 (d, 4H, J ) 8.6 Hz, Ar).
2
Acetylamino-3-butenyl-propanedioic Acid Diethyl Ester. Diethyl
acetamidomalonate, 1.56 g (6.9 mmol), was dissolved at room
temperature under N
2
in 10 mL of dry THF. Potassium tert-butoxide
2
resuspended in 20 µL of distilled H O. Protein expression was
(
0.80 g, 7 mmol) was added under vigorous stirring. The mixture was
monitored by SDS polyacrylamide gel electrophoresis (12% acrylamide
running gel, 12 mA, 14 h), using a normalized OD600 of 0.2 per sample.
Protein Expression (1-L Scale). Similar procedures were used for
preparation and isolation of DHFR from media supplemented with 1,
2, or 3. The example presented is for medium supplemented with 3.
heated for 2 h at 60 °C. 3-Buten-1-ol 4-methylbenzenesulfonate (1.5
g, 6.9 mmol) was added, and the mixture was heated under reflux for
2
days. The THF was removed, the residue was quenched with 10 mL
of 1 M HCl, and the crude product was extracted with ethyl acetate
25 mL). The ethyl acetate solution was washed twice with 25 mL of
water, dried over MgSO , filtered, and concentrated. The crude product
(
4
M9AA medium (100 mL) supplemented with 1 mM MgSO 0.2 wt %
4
glucose, 1 mg/L thiamine chloride, and the antibiotics ampicillin (200
mg/L) and kanamycin (25 mg/L) was inoculated with E. coli strain
CAG18491/pREP4/pQE15 and grown overnight at 37 °C. This culture
was used to inoculate 900 mL of M9AA medium supplemented as
described. The cells were grown to an OD600 of 0.94 and the medium
shift was performed as described for the small-scale experiments,
followed by addition of 40 mL of 1 mg/mL DL-2-amino-5-hexynoic
acid (3). IPTG (0.4 mM) was added, and samples were taken at 1-h
intervals. OD600 was measured, the samples were sedimented and
decanted, and the cell pellets were resuspended in 20 µL of distilled
H O. Protein expression was monitored by SDS polyacrylamide gel
2
electrophoresis (12% acrylamide running gel, 12 mA, 15 h).
Protein Purification. Approximately 4.5 h after induction, cells were
sedimented (9800g, 10 min, 4 °C) and the supernatant was removed.
The pellet was placed in the freezer overnight. The cells were thawed
for 30 min at 37 °C, 30 mL of buffer (6 M guanidine-HCl, 0.1 M
was purified by column chromatography (eluent cyclohexane/ethyl
acetate 2/1 v/v) to yield 0.82 g (44%) of acetylamino-3-butenyl-
1
propanedioic acid diethyl ester. H NMR (CDCl
3
): δ 1.28 (t, 6H, J )
dCHs
), 2.45 (m, 2H, J ) 8.3 Hz, CH
), 4.25 (q, 4H, J ) 7.2 Hz, CH sCH ), 4.90-5.09 (m,
) 10.4, J ) 16.6, Jgem ) 3.2 Hz, CH sCHdCH ), 5.61-5.90
m, 1H, J ) 10.4, J ) 16.6, J ) 6.5 Hz, CH sCHdCH ), 6.78 (s,
H, CONHsCH ).
DL-2-Amino-5-hexenoic acid. The diethyl ester obtained as described
above was hydrolyzed to the dicarboxylate by heating under reflux for
h in 25 mL of 10 w% NaOH. The solution was neutralized with 6 M
HCl, and the solvent was evaporated. The diacid was extracted with
5 mL of methanol. After the solvent was evaporated, 20 mL of 1 M
7
.2 Hz, CH
CH sCH ), 2.08 (s, 3H, CONHsCH
CHsCH sCH
H, J
3 2
sCH ), 1.78-2.0 (m, 2H, J ) 8.3, 6.5 Hz, CH
2
2
2
3
2
d
2
2
3
2
2
Z
E
2
2
(
1
Z
E
2
2
3
4
2
HCl was added and the solution was refluxed for 3 h. The solvent was
evaporated, and the product was taken up in 10 mL of methanol.
Propylene oxide (5 mL) was added, and the mixture was stirred
overnight at room temperature. The precipitate was filtered and dried,
yielding DL-2-amino-5-hexenoic acid (0.47 g, 63%). The product was
recrystallized from EtOH/H
2 4
NaH PO , 0.01 M Tris, pH 8) was added, and the mixture was shaken
at room temperature for 1 h. The cell debris was sedimented (15300g,
20 min, 4 °C), and the supernatant was subjected to immobilized metal
affinity chromatography (Ni-NTA resin) according to the procedure
1
18
2
O 2/1 v/v (0.28 g, 60%). The H NMR
described by Qiagen. The supernatant was loaded on 10 mL of resin
1
data were in agreement with those of ref 16. H NMR (D
2
O): δ 1.78-
), 2.08-2.20 (m,
which was then washed with 50 mL of guanidine buffer followed by
25 mL of urea buffer (8 M urea, 0.1 M NaH PO , and 0.01 M Tris, pH
2 4
2
2
H
3
.0 (m, 2H, J ) 6.4, 6.6 Hz, CH
H, J ) 6.1, 6.4 Hz, CH dCHsCH
NsCHsCOOH), 4.90-5.12 (m, 2H, J
.3 Hz, CH sCHdCH ), 5.61-5.90 (m, 1H, J
6.6 Hz, CH sCHdCH
CH sCH ), 29.9 (CH dCHsCH
16.3 (CH sCHdCH ), 137.3 (CH
Determination of Translational Activity. Buffers and media were
2
dCH-CH
2
sCH
2
2
2
sCH
2
), 3.75 (t, 1H, J ) 6.1 Hz,
8). Similar urea buffers were used for three successive 25-mL washes
at pH values of 6.3, 5.9, and 4.5, respectively. Target protein was
obtained in washes at pH 5.9 and 4.5. These washes were combined
and dialyzed (Spectra/Por membrane 1, MWCO ) 6-8 kDa) against
running distilled water for 4 days, followed by batchwise dialysis against
doubly distilled water for 1 day. The dialysate was lyophilized to yield
70 mg of modified DHFR (DHFR-Y). A similar procedure using
medium supplemented with 2 yielded 8 mg of DHFR-E. A control
experiment in 2× YT medium afforded 60 mg of DHFR. Amino acid
analyses were provided by Dr. C. Cote, University of Massachusetts.
Electrospray mass spectrometry was performed by Dr. W. McMurray,
Yale University School of Medicine. N-terminal protein sequencing
was done by Dr. J. Carlton, Louisiana State University.
2
Z
) 10.5, J
E
) 16.7, Jgem
)
2
2
Z
) 10.5, J
E
) 16.7, J
1
3
)
2
2
). C NMR (D
sCH ), 54.4 (H
sCHdCH ), 174.8 (COOH).
2
2
O): δ 28.9 (CH dCHs
2
2
2
2
2
2
NsCHsCOOH),
1
2
2
2
2
1
7
prepared according to standard protocols. The E. coli methionine
auxotroph CAG18491 (λ , rph-1, metE3079.:Tn10) kindly provided
by the Yale E. coli Genetic Stock Center, was transformed with
plasmids pREP4 and pQE15 (Qiagen), to obtain the expression host
CAG18491/pREP4/pQE15.
-
Protein Expression (5-mL Scale). M9AA medium (50 mL)
supplemented with 1 mM MgSO
4
, 0.2 wt % glucose, 1 mg/L thiamine
Enzyme Purification and Activation Assays. The fully active,
truncated form of methionyl tRNA synthetase (MetRS) was purified
from overnight cultures of JM101 cells carrying the plasmid pGG3.
(The plasmid, which encodes the truncated form of MetRS, was kindly
donated by Professor Hieronim Jakubowski of UMDNJsNew Jersey
Medical School, Newark, NJ.) The enzyme was purified by size
exclusion chromotography as previously described.19 Activation of
methionine analogues by MetRS was assayed via the amino acid-
chloride, and the antibiotics ampicillin (200 mg/L) and kanamycin (25
mg/L) was inoculated with 2 mL of an overnight culture of CAG18491/
pREP4/pQE15. When the turbidity of the culture reached an optical
density at 600 nm (OD600) of 0.8, a medium shift was performed. The
cells were sedimented for 10 min at 3030g at 4 °C, the supernatant
was removed, and the cell pellet was washed twice with 20 mL of M9
medium. Cells were resuspended in 50 mL of the M9AA medium
described above, without methionine. Test tubes containing 5-mL
19,20
i
dependent ATP-PP exchange reaction, also as previously described.
The assay, which measures the 32P-radiolabeled ATP formed by the
(
16) Hatanaka, S.-I.; Furukawa, J.; Aoki, T.; Akatsuka, H.; Nagasawa,
E. Mycoscience 1994, 35, 391.
17) (a) Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular Cloning:
(18) The Qiagen Expressionist, Purification Procedure 7 1992, 45.
(19) Mellot, P.; Mechulam, Y.; LeCorre, D.; Blanquet, S.; Fayat, G. J.
Mol. Biol. 1989, 208, 429.
(20) (a) Blanquet, S.; Fayat, G.; Waller, J.-P. Eur. J. Biochem. 1974,
44, 343. (b) Ghosh, G.; Pelka, H.; Schulman, L. H. Biochemistry 1990, 29,
2220.
(
A Laboratory Manual, 2nd ed.; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, NY, 1989. (b) Ausubel, F. M.; Brent, K.; Kingston, K. E.;
Moore, D. D.; Scidman, J. G.; Smith, J. A.; Struhl, K. Current Protocols
in Molecular Biology; John Wiley & Sons: New York, 1995.