Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451
Weinheim, 2009
DOI: 10.1002/cbic.200900407
Supporting Information
for
Introduction of an Aliphatic Ketone into Recombinan
t Proteins in a
Bacterial Strain that Overexpresses an Editing-Impa
ired Leucyl-tRNA
Synthetase
Yi Tang, Pin Wang, James A. Van Deventer, A. James
Link, and David A. Tirrell*
Experimental Methods
Materials
:
Amino acids
1
and
4
were obtained from Sigma (St. Louis, MO). Amino
acids
2
and
3
were prepared by alkylation of diethyl acetamidoma
lonate with the alkyl
tosylate or alkyl bromide, followed by hydrolysis,
decarboxylation and deacylation.
[1]
Amino acid analogs
2
and
3
were used as the racemates in both in vitro and in
vivo
assays. [
32
P]-radiolabeled sodium pyrophosphate was purchased
from NEN Life Sci-
ence Products, Inc (Waltham, MA). Oligonucleotides
were synthesized at the Caltech
Biopolymer Synthesis Center (Pasadena, CA). General
cloning was performed in XL-
1 blue cells (Stratagene). Expression strain SG1300
9 was obtained from Qiagen.
Synthetase expression and purification
:
E. coli
LeuRS was purified from SG13009
cells transformed with the plasmid p32leus
[2]
(which encodes the synthetase under
the control of an IPTG-inducible promoter), and wit
h the pREP4 repressor plasmid.
Protein expression was induced at
OD
600
= 0.6 by addition of IPTG to a final concen-
tration of 0.5 m
M
. Cells were harvested after 3 hours by centrifugat
ion and lysed by
sonication. The enzyme was isolated using Ni-NTA ag
arose resin under native condi-
tions according to the manufacturer’s instructions
(Qiagen). Proteins were stored in
Buffer A/glycerol (50 m
M
Tris-HCl, 1 m
M
DTT, 50% glycerol). Aliquots were flash
frozen in liquid nitrogen and stored at -80°C. The
concentration of each enzyme was
determined by measuring the absorbance at 280 nm un
der denaturing conditions.
ATP-PP
i
exchange assay
:
Amino acid activation was monitored by an amino aci
d
dependent ATP-pyrophosphate (PP
i
) exchange assay at room temperature
[3]
in buf-
fer containing HEPES (50 m
M
, pH 7.6), MgCl
2
(20 m
M
), DTT (1 m
M
), ATP (2 m
M
),
[
32
P]-PP
i
(0.5 TBq mol
-1
, 2 m
M
), and LeuRS (75 n
M
). The amino acid concentrations
varied depending on the activity of the enzyme towa
rd the substrate. Reactions were
initiated with the addition of LeuRS. Aliquots (20
μL) were quenched using a standard
quench solution (500 μL) containing PP
i
(200 m
M
), HClO
4
(7% w/v), and activated
charcoal (3% w/v). The charcoal was washed twice us
ing a solution of PP
i
(10 m
M
)
and HClO
4
(0.5% w/v), and counted using a scintillation coun
ter. Kinetic parameters
(
k
cat
and
K
m
) were determined by nonlinear regression analysis
of curve-fitting of the
Michaelis-Menten equation. The results reported in
Table 1 are averages from tripli-
cate experiments.
Construction of expression plasmids
: The expression plasmid pA1EL was con-
structed as described by Tang et al.
[2]
It encodes a synthetic leucine zipper protein
A1 under control of an inducible T5 bacteriophage p
romoter and the
E. coli leuS
gene with its endogenous promoter. The mutations at
position T252 of
leuS
were in-
troduced by overlap-extension-PCR mutagenesis using
the Quikchange site-directed
mutagenesis kit (Stratagene) and pA1EL as a templat
e. The primer pairs were used
to generate the tyrosine, isoleucine and valine mut
ations. The integrity of the coding
regions was confirmed through DNA sequencing. The p
lasmids carrying the T252Y,
T252I, and T252V mutants were designated as pA1T252
Y, pA1T252I, and
pA1T252V respectively. Overexpression of LeuRS was
verified by SDS-PAGE of
whole cell lysates from overnight cultures.
Analog Incorporation Assay
: The leucine auxotrophic strain LAM1000
[4]
was trans-
formed with pA1EL, with pA1T252Y (or pA1T252I or pA
1T252V), and with pREP4 to
yield the A1 expression strains LAM1000/(pA1EL, or
pA1T252Y, or pA1T252I, or
pA1T252V)/pREP4. M9 (200 mL) containing 20 canonica
l amino acids (40 mg L
-1
),
MgSO
4
(1 m
M
), CaCl
2
(1 m
M
), glucose (0.4%
w/v
), thiamine (5 mg mL
-1
), ampicillin
(200 mg mL
-1
), and kanamycin (25 mg mL
-1
), was inoculated with an overnight cul-
ture (1 mL) of the expression strain. When the turb
idity of the culture reached an
OD
600
between 0.9 and 1.0, the cells were sedimented and
the cell pellet was
washed with salt solution (0.9% NaCl in H
2
O) three times. The cells were resuspend-
ed in fresh M9 medium with 16 natural amino acids (
M9 –LIVM medium). Leu, Ile, Val,
and Met were not added to the medium (Ile, Val, and
Met were omitted in order to re-
duce background expression levels as previously des
cribed).
[2]
Test tubes containing
aliquots (10 mL) of the resulting culture were prep
ared and supplemented with
2
(100
mg L
-1
),
3
(200 mg L
-1
) or
4
(320 mg L
-1
). Leucine (40 mg L
-1
) was added to a separ-
ate culture (10 mL) as a positive control. A cultur
e without additional amino acids (or
any analog) served as the negative control. Protein
expression was induced by addi-
tion of IPTG to the culture at a final concentratio
n of 1 m
M
. Cultures were grown for 3
h and the cells were collected by centrifugation (5
000g, 10 min, 4°C), resuspended in
Buffer B (600 μL, 8.0
M
urea, 0.1
M
NaH
2
PO
4
, 0.01
M
Tris, pH 8.0) and frozen at
-80°C. Protein expression was monitored by SDS-PAGE
and visualized by Coomas-
sie Brilliant Blue staining.
Protein composition analysis
:
The target protein A1 was purified from cells resus
-
pended in Buffer B (600 μL) by using Ni-NTA spin co
lumns (Qiagen) according to the
manufacturer’s instructions. A1 protein was eluted
in Buffer B (400 μL), pH 4.5. A
portion of the eluent (10 μL) was diluted in ammoni
um bicarbonate (90 μL, 50 m
M
(NH
4
)
2
CO
3
). Trypsin stock solution (5 μL, 0.1 μg μL
-1
) was added, and the sample
was incubated at room temperature overnight. The re
action was quenched by addi-
tion of trifluoroacetic acid (TFA, 2 μL). The react
ion mixture was subjected to C18
ZipTip (Millipore) purification, and peptide fragme
nts were eluted (3 μL) in a solution
of TFA (0.1%) and CH
3
CN (50%). A portion (1 μL) of C18 ZipTip eluent was
used for
tryptic MALDI analysis. The remaining volume of spi
n column eluent (390 μL) was
dialyzed against water extensively and lyophilized
to a fluffy powder. The powder
was sent directly for MALDI mass spectrometry and a
mino acid analysis.
Modification of Ketone Side Chains
: Purified proteins containing ketone function-
alities were dissolved in PBS buffer (200 μL, pH 6.
0) and added to biotin hydrazide
(BH, 20 μL, 5 m
M
) in PBS. Protein/BH mixtures were incubated at roo
m temperature
for 1 to 1.5 h. Reaction solutions were then washed
twice with distilled water using a
buffer-exchange column (Millipore, MWCO = 5 kDa). S
tandard western blotting pro-
cedures were used to identify proteins modified wit
h BH.
References
[1]
J. C. M. van Hest, K. L. Kiick, D. A. Tirrell,
J. Am. Chem. Soc.
2000
,
122
, 1282-1288.
[2]
Y. Tang, D. A. Tirrell,
Biochemistry
2002
,
41
, 10635-10645.
[3]
T. L. Hendrickson, T. K. Nomanbhoy, P. Schimmel
,
Biochemistry
2000
,
39
, 8180-8186.
[4]
Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima
, W. F. DeGrado, D. A. Tirrell,
Angew.
Chem.
2001
,
113
, 2232-2236;
Angew. Chem. Int. Ed.
2001
,
40
, 1494-1496.