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In-vitro Protein Production for Structure
Determination with the Rapid Translation System (RTS)
Ho S. Cho1*, Jeffrey G. Pelton1, Weiru Wang2, Hisao Yokota1, and David E. Wemmer1,2
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA/USA
2 Department of Chemistry, University of California, Berkeley, CA/USA
*corresponding author: [email protected]
1
The goal of the Berkeley Structural Genomics Center is
to determine the structures of all proteins encoded in the
genomes of Mycoplasma pneumoniae and Mycoplasma
genitalium or structural homologs from other organisms.
To achieve this goal, the Berkeley Structural Genomics
Center is developing high-throughput methods for protein expression for use in X-ray and NMR structure
determination. In collaboration with Roche Molecular
Biochemicals, we are investigating the usefulness of the
RTS for in-vitro protein production to generate target
proteins in quantities suitable for structure determination. Very encouraging results have been obtained with
the test protein phosphoserine phosphatase from
Methanococcus jannaschii. The structure of the phosphoserine phosphatase (PSP) from Methanococcus
jannaschii has been determined previously [1]. In this
study, PSP was produced in milligram quantities with the
RTS, and the data collected by X-ray and NMR for structure determination of PSP were comparable with those
of PSP produced in E. coli. Two labeling methods for
NMR employing 15N-algal amino acids and 15N-glycine
were compared.
Materials and Methods
Cloning of PSP-pIVEX
The gene for PSP was excised from a pET-21a construct
[1] and ligated into pIVEX 2.3-MCS (Roche Molecular
Biochemicals) between the Nde 1 and Bam H1 restriction sites. The new construct, PSP-pIVEX, was verified by
PCR and restriction fragment analysis. The PSP-pIVEX
plasmid DNA was amplified in a 1 l LB culture of DH5-α
E. coli cells and isolated with a plasmid maxi kit. The final
concentration of the purified plasmid was approximately
0.37 mg/ml.
Expression of PSP in RTS
The RTS 500 E. coli HY Kit was used to express labeled
protein for NMR. The RTS 500 E. coli Circular Template
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Kit was used to compare the yield to
the RTS 500 E. coli HY Kit. The
Ho S. Cho
lyophilized components of the RTS 500
E. coli HY Kit were reconstituted
according to the product instructions. 15 µg of PSPpIVEX plasmid DNA were used for a 1-ml reaction. The
1-ml reaction was continuously dialyzed against a 10-ml
feeding mixture, containing additional energy sources
and amino acids, in the RTS Instrument set to 30 °C, with
a stirring speed of 120 rpm. After 20 hours of incubation,
a 5-µl aliquot was removed and checked for protein
production on a 15 % SDS-PAGE gel stained with
Coomassie blue. The reaction was stopped at 24 hours,
and the reaction mix was transferred to a 1.5-ml tube
and frozen at –20 °C until purification.
RTS
Introduction
Purification of PSP from a 1-ml RTS reaction
The 1-ml RTS reaction mixture was diluted with 2 ml of
50 mM Tris at pH 7.5 and separated into three 1.5-ml
tubes. The samples were incubated at 70 °C for 30 minutes to precipitate the E. coli proteins. The precipitate
was pelleted by centrifugation at 13,000 rpm for 15 minutes in a microcentrifuge. The supernatant was combined and placed in a dialysis bag with a molecular
weight cut-off (MWCO) of 6 - 8 kD, and dialyzed against
1 l of 20 mM Tris at pH 8.4 for 4 hours. After dialysis, the
sample was passed through a 1-ml Hightrap-Q column
(Pharmacia) equilibrated with 20 mM Tris, pH 8.4. The
column was washed with 5 ml of 20 mM Tris, pH 8.4. The
flow through and wash were collected in 1 ml fractions.
5 µl of each fraction was checked on a 15 % SDS-PAGE
gel with Coomassie staining. Fractions containing PSP
were combined and concentrated to 300 µl in a 4-ml
Ultrafree spin concentrator (Millipore) with a 5-kD
MWCO membrane. The buffer was exchanged twice by
adding 3 ml of a solution containing 20 mM Tris at pH 7.5,
300 mM NaCl, 1 mM EDTA, and 10 mM DTT, and reconcentrating to 300 µl. The sample was then transferred to
a 500-µl Ultrafree spin concentrator and further concentrated to 60 µl. The concentrated protein sample was
used for crystallization.
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Partially Purified
PSP from Material
in Lane 5
E. coli Lysate
(RTS 500 E. coli
HY Kit)
PSP after
Incubation at
30 °C for 20 hours
Partially Purified
PSP from Material
in Lane 3
E. coli Lysate
(RTS 500 E. coli Circular
DNA Template Kit)
Molecular Weight Marker
Seite 28
PSP after
Incubation at
30 °C for 20 hours
M.W
(kD)
16:17 Uhr
E. coli Lysate prior
to Incubation
24.09.2001
Molecular Weight Marker
bc04-11-20.9.rz
50
40
30
20
10
" Figure 1: Coomassie-stained 15 % SDS-PAGE
showing PSP produced in the RTS. Approximately 1 µl of
reaction material was loaded in each lane
RTS
Crystallization and data collection
PSP protein generated in the RTS was crystallized using
the hanging drop vapor diffusion method with seeding in
a buffer that had been utilized previously for crystallization
of PSP produced in E. coli [1]. One µl of concentrated PSP
was mixed with 1 µl of a well solution containing 0.1 M
sodium acetate buffer, pH 4.5, 0.2 M sodium phosphate
dihydrate, 5 mM MgCl2, and 22 % polyethylene glycol
2000 monomethylether (PEG2K MME). Micro-seeding
was performed 1 hour after the drop was set up. Crystals
appeared within 24 hours. The concentration of PEG2K
MME was then raised to 30 % to stabilize the crystals.
Crystals from the drop were flash frozen in liquid nitrogen
and used directly for cryo-crystallography data collection.
X-ray diffraction data were collected at the Advanced
Light Source (ALS) (Berkeley, CA) beam line 5.0.2 using
an Area Detector System Co. Quantum 4 CCD detector
placed 140 mm from the crystal.
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“Uniformly” 15N-labeled PSP
An almost uniformly 15N-labeled PSP sample was generated by using 15N-algal amino acids (Cambridge Isotope
Labs) with the RTS 500 E. coli HY Kit (Roche Molecular
Biochemicals). A stock solution of 15N-labeled amino acids
was prepared by dissolving 100 mg of the algal amino acid
mixture in 6 ml of reconstitution buffer. Because the algal
amino acid mixture is produced from acid hydrolysis of
algal proteins, it does not contain Asn, Cys, Gln, or Trp.
Except for Trp, which does not occur in PSP, 42 mM solutions of the unlabeled forms of the other three amino acids
were used to supplement the 15N-algal amino acids. A
total of 2.805 ml of 15N-algal amino acids stock solution
was combined with 135 µl of Asn, 30 µl of Cys, and 30 µl of
Gln to give a final volume of 3.0 ml. This labeling stock
solution of amino acids was used together with the
RTS 500 E. coli HY Kit according to the product instructions. The same parameters as described above were followed for protein production and purification, except for
the concentration step. After the Hightrap-Q column, the
fractions were pooled and the buffer was exchanged with
a buffer containing 10 mM sodium phosphate at pH 6.5,
10 mM DTT, 20 mM MgCl2, and 0.5 mM EDTA. The protein
was concentrated to a final volume of 450 µl. Fifty µl of D2O
were added to the sample and the pH was adjusted to 6.5
prior to NMR data collection.
15N-Gly
labeled PSP
A 15N-Gly-labeled PSP sample was produced with the
RTS 500 E. coli HY Kit by substituting unlabeled Gly with
15N-Gly (Isotech). The labeling stock solution was
prepared by combining different amounts of 42 mM
" Figure 2: Crystals of PSP generated with protein isolated from a 1-ml
" Figure 3: X-ray diffraction data collected with the
reaction using RTS 500 E. coli HY Kit. A) Picture of the entire crystallization
crystal in Figure 2B. The crystal diffracted to a resolution
drop. B) Close-up view of a crystal suitable for X-ray diffraction with
limit of 1.5 Å, comparable to the resolution limit obtained
dimensions of 100x150x100 µm
with crystals of PSP expressed in E. coli
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1H-15N
labeled with
15N-algal
HSQC spectrum of PSP “uniformly”
amino acids using the RTS 500
E. coli HY Kit. The spectrum was recorded on a Bruker
7
HN ppm
HN ppm
" Figure 4:
N ppm
122
N ppm
118
" Figure 5:
1H-15N
labeled with
15N-Gly
HSQC spectrum of PSP selectively
using the RTS500 E. coli HY Kit. The
spectrum was recorded on a Bruker DRX 500 MHz
AMX 600 MHz spectrometer with the sample temperature
spectrometer with the sample temperature set to 25 °C
set to 25 °C and a total recording time of 17 hours. Peaks
and total recording time of 17 hours. Peaks corresponding
corresponding to Asn, Cys, Gln, and Glu residues are
to 14 of the 15 Gly residues in PSP were observed
missing from the spectrum because the unlabeled forms
of these amino acids were used to supplement the
amino acid mixture
RTS
15N-algal
stock solution of each amino acid depending on the
frequencies of occurrence of these amino acids in PSP.
The volumes in microliter and copy numbers (in brackets) of each amino acid were as follows: 240 Ala (17),
105 Arg (8), 135 Asn (9), 195 Asp (13), 30 Cys (2), 30 Gln
(1), 375 Glu (27), 225 15N-Gly (15), 0 His (0), 300 Ile (22),
270 Leu (20), 450 Lys (33), 45 Met (3), 105 Phe (7), 45 Pro
(3), 75 Ser (5), 105 Thr (8), 0 Trp(0), 45 Tyr (3), 225 Val
(15). The final volume of the amino acid mixture was
3.0 ml. The labeling stock solution was used according to
the product instructions for the RTS 500 E. coli HY Kit.
NMR data collection
1H-15N HSQC spectra [2] were recorded on Bruker AMX
600 MHz and DRX 500 MHz spectrometers. For each
experiment, the sample temperature was set to 25 °C
and the total recording time was 17 hours. The data were
processed using the NMRPipe software suite [3].
(Figure 3). 1H-15N HSQC spectra of 15N-“uniformly”
labeled and 15N-Gly selectively labeled samples of PSP
generated in the RTS show encouraging results for NMR
structure determination (Figures 4 and 5). Except for the
weakest peak, all of the peaks in the 15N-Gly spectrum
(Figure 5) can be superimposed with the corresponding
peaks in the spectrum of the 15N-“uniformly” labeled
sample (Figure 4). If similar results are obtained for a
significant number of target proteins, in-vitro protein production with the RTS should play a major role in structural genomics as well as in structural biology in general.
The authors would like to acknowledge Rosalyn Kim for performing the
activity assays of PSP generated in the RTS System.
References
1. Wang W et al. (2001), Structure Fold Design 9: 65-72.
2. Mori S et al. (1995), J Mag Reson B 108(1): 94-98.
3. Delaglio F et al. (1995), J Biomol NMR Nov, 6(3): 277-93.
Results and Discussion
RTS reactions were easy to set up and produced PSP
consistently in each reaction. A 1-ml reaction with the
RTS 500 E. coli HY Kit yielded 2–3 mg of protein in the
crude reaction mixture and about 1.5 mg of purified PSP
(Figure 1). In addition, PSP protein produced in RTS was
active and could be crystallized using the same conditions for PSP expressed in E. coli [1] (Figure 2). X-ray
crystallography data collected with synchrotron radiation
at the ALS indicated that the crystals produced were of
high quality, diffracting to a resolution limit of 1.5 Å
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Product
Pack Size
Cat. No.
RTS 500 Instrument
1 instrument
3 064 859
RTS 500 E. coli HY Kit
1 kit
(2 reactions)
1 kit
(5 reactions)
3 246 817
1 kit
(for 5 reactions)
3 262 154
RTS 500 Amino Acid
Sampler
3 246 949
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