MINIREVIEW
Wheat germ cell-free platform for eukaryotic protein
production
Dmitriy A. Vinarov, Carrie L. Loushin Newman and John L. Markley
Center for Eukaryotic Structural Genomics, Biochemistry Department, University of Wisconsin-Madison, Madison, WI, USA
Introduction
One of the most important tasks in biotechnology
today is the development of improved systems and
strategies for synthesizing any desired protein or pro-
tein fragment in its folded, soluble form on a prepara-
tive scale. This task is fundamental to the success of
structural genomics projects, which promise to capital-
ize upon numerous advances in science and technol-
ogy to change the appreciation and understanding of
biological systems. Structural genomics implies a
move away from hypothesis-driven research to a sys-
tem of solving structures first and using these struc-
tures and other structures modeled from them as the
source of hypotheses for further research. The medical
incentives for understanding protein structure are
great. Many diseases are caused by defects in a single
protein that alter its folding, stability, or activity. The
structures of proteins involved in diseases will move
us a step closer to improving disease treatment, diag-
nosis, and prevention. Beyond their specific medical
applications, structural genomics projects are teaching
fundamental lessons about the structural basis of life
on this planet.
Keywords
cell-free extract; in vitro; isotopic labeling;
NMR screening; NMR structure

teins are screened by
1
H-
15
N correlated NMR spectroscopy to determine
whether the protein is a good candidate for solution structure determin-
ation. Targets that pass this second screen are then translated in a medium
containing amino acids doubly labeled with
15
N and
13
C. We describe the
automation of these steps and their application to targets chosen from a
variety of eukaryotic genomes: Arabidopsis thaliana, human, mouse, rat,
and zebrafish. We present protein yields and costs and compare the wheat
germ cell-free approach with alternative methods. Finally, we discuss
remaining bottlenecks and approaches to their solution.
Abbreviations
CESG, Center for Eukaryotic Structural Genomics; GST, glutathione S-transferase; HSQC, heteronuclear single-quantum correlation
spectroscopy; IMAC, immobilized metal affinity chromatography; PDB, Protein Data Bank; [U-
15
N]-, uniform labeling with nitrogen-15;
SAIL, stereo-array isotope labeled; Se-Met, selenomethionine.
4160 FEBS Journal 273 (2006) 4160–4169 ª 2006 The Authors Journal compilation ª 2006 FEBS
Protein production remains a bottleneck in proteo-
mics, for both structural and functional studies. Most
structural biology groups and structural genomics cen-
ters utilize cell-based, heterologous protein production
from Escherichia coli. However, this approach fails
with many individual proteins, particularly those from

achieve their native folded state. Platforms for struc-
tural investigations must support the production of
proteins on the scale of 2–10 mg. For efficient struc-
ture determination by NMR spectroscopy, the proteins
must be labeled with stable isotopes (
15
Nor
13
C+
15
N,
or for larger proteins
2
H+
13
C+
15
N). For X-ray crys-
tallography, proteins normally are labeled with sele-
nomethionine (Se-Met) to support multiwavelength
anomalous dispersion data collection for phase deter-
mination. Because protein production and labeling on
this scale is expensive, it is important to screen targets
first on a smaller scale to identify which constructs are
expressed, soluble without aggregation, folded, and
stable under the conditions used for NMR structure
determinations or crystallization trials.
In vitro cell-free methods for protein synthesis with
extracts from prokaryotic [1] or eukaryotic [2] cells
offer an alternative to the E. coli cell-based platforms.

15
N]-labeled
proteins prior to isolation from the cell-free protein
synthesis mixture [16,17]. One of the features of cell-
free protein production is that only the protein of
interest is labeled, so that contaminating proteins do
not show up in normal multinuclear NMR spectra.
Cell-free protein production protocols are streamlined
compared to cell-based protocols, in that they do not
require cell harvesting or cell lysis. Protein purification
is usually simpler, because the protein of interest starts
out more concentrated and is isolated from a smaller
set of contaminants.
The RIKEN Structural Genomics Center in colla-
boration with Roche has pioneered the use of cell-free
protein production through a coupled transcription-
translation system employing E. coli extracts [18–22].
It has been found, however, that most of the pro-
teins that produce well in E. coli cell-free systems are
the same ones that are produced successfully from
E. coli cells [10]. Thus, despite other potential advan-
tages, the E. coli cell-free approach may not greatly
expand the range of proteins that can be produced in
soluble, folded state, although it may be possible to
overcome this limitation by redesigning the gene
sequence (see below), by adding chaperones or other
factors [22,24], or by reengineering ribosomal proteins
[25].
D. A. Vinarov et al. Wheat germ cell-free eukaryotic protein production
FEBS Journal 273 (2006) 4160–4169 ª 2006 The Authors Journal compilation ª 2006 FEBS 4161

of a plasmid used for in vitro transcription, (2) small
scale (25–50 lL) screening to assay the level of protein
production and solubility, (3) larger scale (4–12 mL)
production of [U-
15
N]-protein used to evaluate whe-
ther solution conditions can be found that render the
target suitable for NMR structure determination
(soluble, monodisperse, folded, and stable), and (4)
production of sufficient [U-
13
C,
15
N]-protein for multi-
dimensional, multinuclear magnetic resonance data
collection. We purchase the wheat germ extract from
CellFree Sciences, Inc., the RNA polymerase from
Promega (Madison, WI), and the labeled amino acids
from Cambridge Isotope Laboratories, (Andover,
MA). Details about these and other reagents and sup-
plies are found in our publications [32–34].
The purification workflow diagram is shown in
Fig. 1B. In step (1), a defined series of cloning proce-
dures are used to create a DNA plasmid containing
the target gene and 5¢ and 3¢ extensions that promote
efficient transcription and translation. In step (2), small
scale protein expression and purification trials are car-
ried out, generally in a 96 well format. Successful can-
didates from these screens (those estimated to yield
> 0.5 mgÆmL

Concentrate
PreScission
Protease Cleavage
2nd GSTrap
Column
Protein product
with cleavable
N-GST tag
Protein product
with non-cleavable
N-(His)
6
tag
Ni-HiTrap
Chelating Column
Concentrate
Superdex75 in
NMR Buffer
Concentration
NMR sample
Cell Free
Reaction
(4-12ml)
Target selection
AB
Screening (50 µl scale)
Analysis, Expression level, Solubility, (Tag cleavage)
3. Production and analysis of [
15
N]-protein

15
N]protein for structure
determination. (B) Schematic illustration of
the steps involved in isolating and purifying
proteins produced by wheat germ cell-free
platform depending on the type of tag: non-
cleavable N-(His)
6
tag or cleavable N-GST
tag.
Wheat germ cell-free eukaryotic protein production D. A. Vinarov et al.
4162 FEBS Journal 273 (2006) 4160–4169 ª 2006 The Authors Journal compilation ª 2006 FEBS
We have tested the wheat germ cell-free platform in
the context of NMR-based structural genomics of
eukaryotic proteins and have compared it with our
parallel E. coli cell-based platform. Our experience is
summarized briefly as follows. (a) Targets can be
screened more quickly and more economically for pro-
tein expression and solubility by the cell-free approach
than by the cell-based approach. The efficiency of this
process is important, because we need to screen many
targets or multiple constructs of a given target in order
to find one that produces a protein that is soluble and
well folded. As an example of multiple screening of a
given target, we have screened targets with a noncleav-
able His
6
tag, with a cleavable His
6
tag, and with a

15
N, selective labeling by residue type, and SAIL
(discussed above).
We recently carried out a detailed comparison of the
wheat germ cell-free and E. coli cell-based approaches
to protein production for NMR structure determin-
ation [35]. In this study 96 randomly chosen Arabidop-
sis thaliana targets were carried through CESG’s wheat
germ cell-free and E. coli cell pipelines. If possible,
[
15
N]-labeled versions of each protein were produced
for analysis by
1
H-
15
N correlation NMR spectroscopy.
Of the 96 targets started with, only eight from the cell-
free pipeline and five from the cell-based pipeline were
found suitable for NMR structural analysis on the
basis of the NMR results. In this comparison, the five
targets that proved successful by the E. coli cell-based
approach also were successful by the cell-free
approach.
Our wheat germ cell-free approach appears to have
advantages over published in vitro protein production
protocols that utilize E. coli S30 extract. (a) Cell-free
protocols utilizing E. coli extract usually call for the
testing of multiple plasmids with sequence differences
outside the protein coding region to determine one

Automation
All of the cell-free operations can be carried out by
hand, and this is how we started using the technology.
Because of the small volume requirements for screen-
ing (25–50 lL) and protein production for structural
studies (4–12 mL), cell-free methods have proved
amenable to automation. CESG makes use a CellFree
Sciences GeneDecoder1000
TM
robotic system (Fig. 2)
in automating the small scale screening of constructs
for protein production and solubility. This unit makes
it possible to carry out as many as 1052 small scale
(25 lL) screening reactions per week. CESG has
two prototype robotic units developed by CellFree
Sciences for larger scale protein production (Fig. 2).
The Protemist10
TM
robotic system requires preparation
of the mRNA off-line, whereas the newer Prote-
mist100
TM
starts with DNA and produces the mRNA
transcript prior to the translation step. Each of these
systems supports 24 4 mL transcription and translation
reactions per week. Typical yields for the Protemist
runs are 0.3–0.5 mg purified protein per mL reaction
mixture. These robotic systems handle the many steps
that are tedious to carry out by hand, and work
D. A. Vinarov et al. Wheat germ cell-free eukaryotic protein production

rescuing failed targets by truncating the N- and ⁄ or
C-termini. The second screening operation relevant to
NMR structure determinations is the screening of the
[
15
N]-labeled protein target by
1
H-
15
N HSQC spectro-
scopy). This test, which is repeated after one week to
Fig. 2. Fully automated protein synthesizers from CellFree Sciences. (Left) GeneDecoder1000
TM
, which operates in two small scale modes.
In the screening mode, it handles up to four 96 well plates per overnight run, produces 2–10 lg protein per well, and uses 1.0–5.0 mL
wheat germ extract per plate. In the small scale protein production mode it can handle up to two 96 well plates per overnight run, produces
between 10 and 50 lg protein per well, and uses 5.0–10.0 mL wheat germ extract per plate. (Center) Protemist10
TM
robotic system, which
is capable of carrying out 24 4 mL translation reactions per week. The unit produces 1–3 mg protein per reaction and utilizes 3 mL wheat
germ extract per reaction. This system requires off-line preparation of the mRNA. (Right) Protemist100
TM
robotic system, which supports
24 4 mL transcription and translation reactions per week. Its capabilities are similar to those of the Protemist10, but it has the added feature
of automated production of mRNA. These robotics systems carry out a variety of operations including solvent extraction, high level multi-
channel liquid handling, centrifugation, and incubation at various temperatures. An onboard microprocessor interfaced with the computer
connected to the database keeps detailed log files that contain information about temperatures, volumes, and operational performance at
every step.
Wheat germ cell-free eukaryotic protein production D. A. Vinarov et al.
4164 FEBS Journal 273 (2006) 4160–4169 ª 2006 The Authors Journal compilation ª 2006 FEBS

proteins fused to GST can be more highly soluble and
that the advantage may persist after the tag is removed
(presumably through improved folding of the purified
fusion protein prior to cleavage).
We have gathered statistics specific to human pro-
teins. Of 174 human targets (most with unknown func-
tion) that were successfully cloned, 135 (78%) showed
expression at levels suitable for structural investiga-
tions. Of these expressed proteins, 55 (41%) were
soluble at levels needed for NMR spectroscopy. Of
these, 36 (66%) gave [
15
N]-labeled samples at levels
that could be evaluated by NMR spectroscopy. To
date, nine of these human proteins yielded NMR
structures. In total, CESG has determined NMR struc-
tures of 18 eukaryotic proteins produced by this meth-
odology (Fig. 3). The average yield of purified, labeled,
human proteins made for NMR structural studies has
been 0.3 mgÆmL
)1
reaction mixture.
Costs
Labor savings, coupled with the high level of incorpor-
ation of labeled amino acids and the high yield of folded
protein samples, makes the overall cost of the wheat
germ cell-free method comparable to that of the E. coli
cell-based approach for NMR structure determinations
of eukaryotic proteins. One of the main advantages of
the automated wheat germ cell-free protein expression

cloned
successfully
Targets
showing
acceptable
expression
Targets
showing
adequate
solubility
[
15
N]-labeled
proteins
produced
Acceptable
[
15
N-
1
H]-HSQC
spectrum
Protein
stable
for >
10 days
[
13
C,
15

12 kDa
PDB: 2FB7
(7) At3g51030.1
14 kDa
PDB: 1XFL
(8) Hs.102419
13 kDa
PDB: 1ZR9
(9) Hs.157607
14 kDa
PDB: 2ETT
(10) At2g23090.1
9 kDa
PDB: 1WVK
(11) P62627
dimer 22 kDa
PDB: 1Y4O
(12) At2g46140.1
19 kDa
PDB: 1YYC
(1) Hs.78877
11 kDa
PDB: 2G2B

(2) At5g39720.1
19 kDa
PDB: 2G0Q
(3) At5g66040.1
14 kDa
PDB: 1TQ1

costs of stable isotope labeled amino acids also may be
expected to decrease as demand accelerates. Average
supplies costs currently are: US$47 per target for clo-
ning and expression solubility testing (with unpurified
reaction mixture assayed by SDS⁄ PAGE), US$370 per
mg for Se-Met protein, US$390 per mg for [
15
N]pro-
tein, and US$470 per mg for [
13
C,
15
N]protein (with
proteins isolated and purified).
The major advantages of the wheat germ cell-free
method over the E. coli cell-based pipeline are that it
supports the production of a larger fraction of targets
as folded, soluble protein and that it is much faster to
prepare additional samples or truncated samples as
needed for successful structure determinations. The
E. coli approach has a cost advantage when its protein
yields are much higher than cell-free. The overall costs
of each approach appear to be similar for NMR struc-
ture determinations.
Prospects
Because of the complementarity of cell-free and cell-
based methods, we envision that it will be most
efficient to screen each new target by both methods.
Initially, we did not have an easy way to screen tar-
gets by the two approaches, because the cell-based

fusions, or different vectors and hosts) and choose the
one that yields the most soluble protein. We have initi-
ated a pilot study aimed at determining whether the
initial production of constructs with multiple N- and
C-termini for small scale screening would be more effi-
cient than our current approach of redesigning failed
constructs.
Currently, CESG’s X-ray structure pipeline requires
in the order of 10 mg of Se-Met protein for each tar-
get. We anticipate that as reliable small scale crystal-
lization screening methods become available, the wheat
germ cell-free method could become part of the X-ray
crystallography pipeline. We have already determined
by mass spectrometry that the wheat germ cell-free
approach supports high level incorporation of Se-Met,
and we have made small quantities of Se-Met-labeled
proteins for use chip (Fluidigm, South San Francisco,
CA) crystallization screening.
Acknowledgements
We gratefully acknowledge the work of all CESG staff
members and collaborators and fruitful interactions
with Professor Y. Endo and his group at Ehime Uni-
versity, Matsuyama, Japan, and staff members of
CellFree Sciences Co., Ltd. (Yokohama, Japan)
in adapting their technology to research and product-
ion environments. Supported by NIH grants 1U54
G074901 (which supports CESG), and P41 RR02301
(which supports the National Magnetic Resonance
Facility at Madison, where NMR spectroscopy was
carried out).

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