Hands-on Demonstration of Wheat Germ
Cell-free Translation
Shin-ichi Makino, Michael A. Goren, Yuko Matsubara, Kazuyuki Takai,
Hidenori Hayashi, Brian G. Fox, John L. Markley and Yaeta Endo
Center for Eukaryotic Structural Genomics
University of Wisconsin-Madison, Madison WI USA
Cell-Free Science and Technology Research Center
Ehime University, Matsuyama Japan
Your cell-free translation kit
Steps to assemble your translation reaction
Flowchart for CESG structural studies
Notes
1. All glassware must be baked for 3 h at 180˚C to eliminate
contaminating RNase.
2. In order to prevent RNase contamination from hands or saliva, wear
gloves and keep the mouth closed while handling reagents.
3. All the buffers must be sterilized by passage through a 0.2 μm filter,
and stored at -20˚C unless otherwise indicated.
4. Plasmid DNA prepared by commercially available kits often contains a
contamination of RNase. This contaminant must be removed for
successful transcription and translation.
5. Contaminating wheat germ extract bands in the first IMAC purification
step can be diminished by use of WEPRO2240H wheat germ extract,
which has been pretreated with an IMAC resin to remove these
endogenous contaminants.
Primer design
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Use a two-step PCR to create
needed gene functionality
1st PCR primers are gene
specific; 2nd PCR primers are
universal
Match the 5’ of gene starting
with 3rd codon of the ORF;
append the TEV protease site
and SgfI site in order to liberate
Ser as N-terminus
Match 3’ of gene with stop
codon; append PmeI site
Plasmid
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SP6 promoter
TMV Omega sequence
sacB-CAT toxic selection
pF1K homology region
CESG plasmids are
available in the PSI-MR
• Use Flexi Vector cloning
• SEQUENCE-VERIFY YOUR
GENE!!!!
Plasmid preparation
Steps
Commentary
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Transformed cells grown in 2YT
Marligen high purity maxi-prep kit
Proteinase K treatment of partially
purified plasmid to remove all
residual RNAse activity
Phenol:chloroform extraction to
remove proteinase K
Acetate/ethanol precipitation
Carefully dry the DNA pellet
Re-suspend in 18 MΩ water; use
A260 measurements to prepare 1
µg/µL
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Plasmid DNA is a valuable
reagent for the transcription
reaction
You need more quantity and
higher purity than for cell-based
cloning
Ethanol and and precipitants are
deleterious
Reagents for transcription
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Transcription Buffer (TB+Mg, 5) is 400 mM HEPES-KOH, pH 7.8,
containing 100 mM magnesium acetate, 10 mM spermidine
hydrochloride, and 50 mM DTT. Store this buffer at -20˚C.
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An NTP solution containing 25 mM each of ATP, GTP, CTP, and UTP
is prepared from 0.2 μm filter-sterilized, 100 mM solutions of each NTP
prepared in Milli-Q water. NTP solutions are stored at -80˚C.
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SP6 RNA polymerase and RNase inhibitor (RNasin) are from Promega.
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The transcription mixture (TB, 2) contains 2 TB+Mg, 8 mM NTPs, 3.2
unit/μL of SP6 RNA polymerase, and 1.6 unit/μL of RNasin. This
solution is prepared immediately before use.
Transcription reaction
Steps
Commentary
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If the transcription reaction is
proceeding correctly, a white
precipitate of magnesium
pyrophosphate will form and make the
transcription solution turbid.
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In order to avoid co-precipitation of
mRNA, the reaction should not be
chilled.
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In a 96-well PCR plate, dispense 2.5
µL of the transcription mixture into
each well and add 2.5 μL of each
separate plasmid DNA prepared as
before to separate wells of the PCR
plate.
Tightly cap the wells to avoid
concentration of the samples by
evaporation. Incubate the transcription
reaction at 37˚C for 4 h.
This RNA solution is used in
translation reactions without further
purification.
Wheat germ extract and proteins
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The wheat germ cell-free extract is WEPRO2240 from CellFree Sciences, Ltd.
(Yokohama, Japan). This preparation has 240 OD260 per mL and is prepared
without amino acids. Store the extract at -80˚C.
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Contaminating wheat germ extract bands in the first IMAC purification step can
be diminished by use of WEPRO2240H wheat germ extract, which has been
pretreated with an IMAC resin to remove these endogenous contaminants.
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Creatine kinase is from Roche Applied Sciences (Indianapolis, IN). Dissolve in
Milli-Q water to make 50 mg/mL and store at -80˚C. Dilute the stock solution to 1
mg/mL prior to use.
Dialysis buffer (substrates)
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Unlabeled amino acids are from Advanced ChemTech (Louisville, KY)
15N and 13C,15N-labeled amino acids are from Cambridge Isotope Laboratories
(Andover, MA).
A mixture of the 20 unlabeled amino acids is prepared in Milli-Q water with each
amino acid present at 2 mM. The 15N-labeled amino acids are prepared at 8
mM, and the 13C,15N-labeled amino acids are prepared at 5 mM, also in Milli-Q
water. Do not filter these preparations because some amino acids are not
dissolved in these solutions.
Dialysis Buffer (DB, 5) is prepared from 120 mM HEPES-KOH, pH 7.8, and
contains 500 mM potassium acetate, 12.5 mM magnesium acetate, 2 mM
spermidine hydrochloride, 20 mM DTT, 6 mM ATP, 1.25 mM GTP, 80 mM
creatine phosphate, and 0.025% (w/v) sodium azide. Store this buffer at -80˚C.
An aliquot of 1 DB containing amino acids is prepared by dilution of 5 DB with
Milli-Q water and addition of the appropriate amino acid mixture to 0.3 mM.
Sonicate the mixture for 5 min, and then pass the solution through a 0.2 µm
filter.
Cofactors, metals, other additives
• Metal content is shown
at right; may require
supplementation
• Cofactors are depleted
relative to level of
protein translation
• Not much lipid present
• Detergents can be
added; we prefer using
liposomes
Small-scale translation reaction
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Prepare the translation mixture. Each individual translation mixture
consists of 6.25 μL of water, 2.75 μL of 5 DB, 3.75 μL of 2 mM
unlabeled amino acids, 1 µL of 1 mg/mL creatine kinase, and 6.25 μL
of WEPRO2240 wheat germ extract.
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Depending on the number of separate reactions to be performed, scale
these volumes and include ~10% extra volume in order to fill each
translation well and account for handling losses.
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Add 125 μL of 1 DB with amino acids to a well of a microtitre plate.
Mix 20 μL of translation mixture with 5 µL of transcription reaction to
make 25 μL reaction. Execute a bi-layer method translation by injecting
the 25 µL reaction under the 1 DB with amino acids. Continue the
reaction for 20 h at 26˚C without disturbing the bi-layer.
Translation
screening—SDS PAGE
SDS-PAGE analysis of proteins (A-H) soluble (S) and insoluble (P)
fractions of expressed proteins. The expressed proteins are marked with an
arrowhead.
The amount of total protein expressed is estimated by visual inspection and
comparison to the intensity of the 31 kDa marker band in the molecular
weight standards, which is present at 0.15 µg. An H total expression rating
corresponds to greater than 2.5 µg of protein expressed per translation
reaction; an M total expression rating corresponds to more than 1.25 µg but
less than 2.5 µg of protein expressed per translation reaction; a W total
expression rating corresponds to less than 1.25 µg of protein expressed per
translation reaction, but still detectable; and a U rating corresponds to an
uncertain determination, possibly because of no detectable expression or
overlap with endogenous wheat germ protein bands. The solubility is
assessed by comparison of the intensity of the corresponding protein bands
in the soluble and insoluble translation products gels.
A high solubility rating (H) is assigned when the soluble translation band
has 3 or greater of stained intensity than the insoluble translation band.
A medium solubility rating (M) is assigned when the soluble translation
band has approximately equal stained intensity with the insoluble
translation band.
A weak solubility rating (W) is assigned when the soluble translation band
has less stained intensity than the insoluble translation band, and an
uncertain solubility rating (U) corresponds to an uncertain determination,
possibly because of no detectable expression or because of overlap with
endogenous wheat germ protein bands.
The proteins investigated, identified by their Gene Ontology
numbers, and their purification ratings are as follows:
A, GO.74329, expression rating H, solubility rating H; B,
GO.34351, expression rating H, solubility rating W; C,
GO.70653, expression rating H, solubility rating W; D, GO.7312,
expression rating H, solubility rating M; E, GO.24674, expression
rating H, solubility rating H; F, GO.79368, expression rating M,
solubility rating M; G, GO.37540, expression rating H, solubility
rating W; H, GO.80048, expression rating H, solubility rating M.
Purification
screening—His tag
SDS-PAGE analysis of proteins purified
from the small-scale cell-free translation
reaction used to screen for protein
production, solubility, and likelihood of
success in large-scale purification.
The two proteins observed in all lanes
(~50 kDa, marked with black dots) are
endogenous wheat germ proteins that copurify with His6-tagged proteins using
IMAC (See Note 5).
The successfully purified protein of
sample A is marked with a white star to
show an example.
The Gene Ontology numbers are the same as in the legend of
Translation Screening—SDS PAGE. The purification ratings are
H, H, U, H, H, M, W, and H (from left to right).
An H rating corresponds to a purified yield of greater than 2.5 μg
per small-scale translation reaction; an M rating corresponds to
a purified yield of 2.5 -1.25 μg per small-scale translation
reaction; a W rating corresponds to a purified yield of less than
1.25 μg per small-scale translation reaction; and a U rating
corresponds to no purified protein detected.
Purification screening—membrane proteins
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Soybean total extract (20% lecithin) is from Avanti Polar Lipids (Alabaster, AL).
The lipid rehydration buffer is 25 mM HEPES, pH 7.5, containing 100 mM NaCl.
Track-etch polycarbonate membranes, 0.4 µm and 0.1 µm, are from Nucleopore
(Pleasanton, CA).
Liposomes are prepared by extrusion.
Accudenz is from Accurate Chemical and Scientific (Westbury, NY). Prepare 80% (w/v) and
35% (w/v) solutions in 25 mM HEPES, pH 7.5, containing 100 mM NaCl and 10% (w/v)
glycerol. Store these solutions at room temperature.
Large-scale translation reaction, part 1
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Prepare the plasmid DNA as described earlier. Plasmid DNA prepared by
commercially available kits often contains a trace contamination of RNase. This
contaminant must be removed for successful transcription and translation.
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To prepare sufficient mRNA for a large-scale protein production, carry out a
transcription reaction in a 50 mL conical tube with a total volume of 4 mL of 1
TB+Mg containing 4 mM NTPs, 0.05 mg/mL of plasmid DNA, 0.5 unit/μL of SP6
RNA polymerase, and 0.25 unit/μL of RNasin. Incubate the reaction at 37˚C for
3 to 5 h. If the transcription reaction is proceeding correctly, a white precipitate
of magnesium pyrophosphate will form and make the transcription solution
turbid.
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Remove the white precipitate from the transcription reaction by centrifugation in
the C0650 rotor and Allegra X-22R centrifuge for 5 min at 6230 rpm (4000  g)
and 26˚C. Transfer the supernatant to a new tube. This clarified solution is used
as the mRNA solution. In order to avoid co-precipitation of mRNA, the reaction
should not be chilled.
Large-scale translation reaction, part 2
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Add 1452 μL of 1 DB with amino acids, 48 μL of creatine kinase (50 mg/mL in
water), 1000 μL of WEPRO2240, and 1500 μL of mRNA solution to an Amicon
Ultra-15 (10 K MWCO) concentrator. Spin the concentrator in the C0650 rotor
and Allegra X-22R centrifuge for 8 min at 5395 rpm (3000  g) and 26˚C. Add 2
mL of 1 DB with amino acids, mix gently by pipetting, and spin for 5 min. Add 1
mL of 1 DB with amino acids, mix gently by pipetting, and spin for 8 min. If the
volume is less than 4 mL, add 1 DB with amino acids to achieve a reaction
volume of 4 mL.
Prepare an exchanging buffer by mixing 50 mL of 1 DB with amino acids and
2.5 mL of mRNA solution. Add sufficient buffer (typically 2.5 mL) to bring the
reaction volume to 6.5 mL. Centrifuge the concentrator tube as above to
decrease the volume to ~4 mL, and incubate at 26˚C for 1 h. Add fresh buffer to
bring the volume back to 6.5 mL and repeat the centrifugation step. This cycle is
repeated 18 times.
The final yield of soluble protein from the large-scale preparation typically
ranges from 0.2 to 0.7 mg of purified protein per 4 mL reaction for a protein
rated H in the small-scale purification trial.
Robots in use at CESG
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A skilled operator can carry out all of the reactions described above by
hand and achieve excellent results. The following robots are used at
CESG in more extensive cell-free translation studies
GeneDecoder1000™
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Protemist100™
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Fully automated transcription and translation at 10˚C to 40˚C. Capable of yielding ~10
µg of protein in each of the 384 (4 x 96) wells in 24 h, with a 4 h transcription step and
20 h translation cycle. A typical reaction volume is 25 µL.
Large-scale synthesis robot with fully automated transcription and translation. Capable
of up to 8 translation reactions in a fully automated mode over a 24-h period. In our
experience, 4 mL reactions yield an average of 0.5 mg protein.
DT-II™
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Bench-top robot capable of fully automated screening or production. Screening mode
carries out 24 x 1.2 mL reactions for 24 h. Production mode carries out 6 x 6 mL
reactions with yield of ~1 mg per run. DT-II will perform automated purification of Hisor GST-tagged proteins, including treatment with either PreScission or TEV protease to
remove tags as part of the automation cycle.
Summary
• Wheat germ cell-free translation gives rapid access
to proteins for functional and structural studies
• Care with reagents is needed to assure success
• Scale-up of the production of proteins identified by
small-scale screening is proportional to the volume
increase
• Open system provides options for addition of metal
and cofactors, and co-translation to produce multiprotein complexes
• Membrane proteins are translated with liposomes in a
functional state and easily purified
Acknowledgements
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This work was supported by NIGMS Protein Structure Initiative grant
1U54 GM074901 (J.L. Markley, PI, G.N. Phillips and B.G. Fox, CoInvestigators, which supports CESG), NIGMS grant P41 RR02301 (J.L.
Markley, PI, which supports the National Magnetic Resonance Facility
at Madison, where NMR spectroscopy was carried out), and NIGMS
50853 (B.G. Fox, PI). M.A. Goren was recipient of an NSF East Asia
and Pacific Summer Institutes Fellowship. Dr. Dmitriy A. Vinarov led
initial work at CESG on the development of the wheat germ cell-free
system for structural genomics efforts with assistance from Ms. Ejan
Tyler and Dr. Carrie Loushin Newman.
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The authors thank Prof. Yaeta Endo and others at Ehime University for
advice and encouragement and also the staff members of CellFree
Sciences Ltd. (Yokohama, Japan) who made this approach
commercially viable.
References
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FlexiVector Cloning
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Cell-free translation—screening
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Tyler, R. C., D. J. Aceti, C. A. Bingman, C. C. Cornilescu, B. G. Fox, R. O. Frederick, W. B. Jeon, M. S. Lee, C. S. Newman, F. C.
Peterson, G. N. Phillips, Jr., M. N. Shahan, S. Singh, J. Song, H. K. Sreenath, E. M. Tyler, E. L. Ulrich, D. A. Vinarov, F. C. Vojtik,
B. F. Volkman, R. L. Wrobel, Q. Zhao, and J. L. Markley. 2005. Comparison of cell-based and cell-free protocols for producing
target proteins from the Arabidopsis thaliana genome for structural studies. Proteins 59:633-43.
Vinarov, D. A., B. L. Lytle, F. C. Peterson, E. M. Tyler, B. F. Volkman, and J. L. Markley. 2004. Cell-free protein production and
labeling protocol for NMR-based structural proteomics. Nat Methods 1:149-53.
Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput
structure determination. Draft provided for instructional purposes.
Cell-free translation—production for structural studies
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Blommel, P. G., P. A. Martin, R. L. Wrobel, E. Steffen, and B. G. Fox. 2006. High efficiency single step production of expression
plasmids from cDNA clones using the Flexi Vector cloning system. Protein Expr Purif 47:562-70.
P. G. Blommel, P. A. Martin, K. D. Seder, R. L. Wrobel and B. G. Fox. (2007). Flexi Vector cloning in high throughput protein
expression and purification. In Methods in Molecular Biology (S. Doyle, Ed.), The Humana Press Inc., Totowa, N.J.
Vinarov, D. A., C. L. Newman, E. M. Tyler, J. L. Markley, and M. N. Shahan. 2006. Wheat germ cell-free expression system for
protein production. Curr Protoc Protein Sci Chapter 5:Unit 5 18.
Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput
structure determination. Draft provided for instructional purposes.
Membrane proteins, liposomes density gradient purification
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Goren, M. A., and B. G. Fox. 2008. Wheat germ cell-free translation, purification, and assembly of a functional human stearoylCoA desaturase complex. Protein Expr Purif 62:171-78.
Makino, S., M.A. Goren, B.G. Fox, and J.L. Markley (2009) Cell-free protein synthesis technology in NMR high-throughput
structure determination. Draft provided for instructional purposes.
Hands-on Demonstration of Wheat Germ
Cell-free Translation
Shin-Ichi Makino, Michael A. Goren, Yuko Matsubara, Kazuyuki Takai,
Hidenori Hayashi, Brian G. Fox, John L. Markley and Yaeta Endo
Center for Eukaryotic Structural Genomics, University of Wisconsin-Madison, Madison WI
USA and Cell-Free Science and Technology Research Center, Ehime University Ehime
University, Matsuyama Japan
Steps to assemble your translation reaction
Time lapse photos of translation reaction
SDS-PAGE analysis of translation reaction