Download Rapid Translation System RTS 500 E. coli HY Kit

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Rapid Translation System RTS 500 E. coli HY Kit –
the Second Generation Cell-free System
Expressing Milligram Amounts of Protein
Erhard Fernholz*, Manfred Watzele, Claudia Kirr, and Bernd Buchberger
Roche Molecular Biochemicals, Penzberg/Germany
*corresponding author: [email protected]
Cell-free protein expression has only been used as an analytical tool because of the low protein
yields that could be obtained. With the Rapid Translation System RTS 500 HY it is now
possible to express milligram amounts of protein in less than 24 hours. It also enables researchers
to label proteins with high specificity and cost-effectiveness for X-ray and NMR analysis.
Introduction
RTS
After the completion of the human genome project,
numerous structural genomics initiatives were launched to
promote the basic understanding on how nature works at
the protein level. For this purpose, there is a demand for
rapid protein expression systems which guarantee high
yields and are convenient and amenable to automation.
Cell-free protein expression has a strong potential to meet
these requirements because handling of intact cells (e.g.,
transformation, fermentation, lysis) is not necessary. In
fact, this methodology only involves liquid handling and
can thus be adapted to automated processes.
Low yield has been the limiting factor for many years in
cell-free protein expression making this method suitable
only as an analytical tool. The development of the RTS 500
E. coli Circular Template Kit in the year 2000 has marked
the beginning of preparative scale, cell-free protein expression. The newly developed RTS 500 E. coli HY Kit now
enables expression of up to 5 mg protein/ml within a day.
This kit uses the Continuous Exchange Cell-Free (CECF)
Table 1: Features of available RTS 100 and RTS 500
platforms
Feature
CECF-technology
HY biochemistry
Standard reaction
volume
RTS 500
Instrument
Standard
reaction time
Protein yield
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BIOCHEMICA · No. 4 · 2001
RTS 100
E. coli
HY Kit
RTS 500
E. coli
HY Kit
no
yes
50 µl
yes
yes
1 ml
RTS 500
E. coli
Circular
Template Kit
yes
no
1 ml
not
required
2 to 4 h
required
required
24 h
24 h
up to
400 µg/ml
up to
5 mg/ml
up to
500 µg/ml
technology and is based on the RTS 500 E. coli Circular
Template Kit with improved reaction conditions (Table 1).
Together with the recently introduced RTS 100 E. coli HY
Kit for small scale protein expression reactions, this new kit
will enable researchers to speed up their gene-to-protein
experiments. Since the amino acids are now separated
from the reaction and feeding mixtures, the design of the
kit allows the incorporation of labeled amino acids.
Materials and Methods
The Rapid Translation Systems RTS 100 E. coli HY, RTS
500 E. coli Circular Template and RTS 500 E. coli HY were
operated according to the pack insert. In case of Green
Fluorescent Protein (GFP) expressions, the reaction
chamber was half-filled to allow cotranslational oxidative
maturation. In general, the synthesized protein was
Reaction
Chamber Stir Bar
Membrane
• DNA Template
• T7 RNA Polymerase
• Translation Machinery E. coli
• Energy Regenerating System
Inhibitory
By-Products
Supply
• Amino Acids
• Energy Substrates
• Buffer Components
• NTPs
Feeding
Chamber
Stir Bar
" Figure 1: Continous Exchange Cell Free (CECF)
protein expression with the RTS 500. The RTS 500 device
is composed of a reaction chamber, a semi-permeable
membrane, and a feeding chamber. Transcription
and translation take place simultaneously in the
reaction chamber
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β-Galactosidase
(116 kD)
Green Fluorescent
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ErythropoetinThioredoxin Fusion
Protein (33 kD)
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Erythropoetin
(22 kD)
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Seryl tRNA
Synthetase (64 kD)
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Protein 1049
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(30 kD)
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# Figure 2: Comparison of
the RTS 500 E. coli HY Kit and the
RTS 500 E. coli Circular Template Kit.
Proteins of different size and nature
were expressed in the HY Kit (a) and
the Circular Template Kit (b)
GFP/Methionine (M = 149.2 g/mol) GFP/Selenomethionin (M = 196.1 g/mol)
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Intensity (cps)
For seleno-methionine labeling, the methionine in the
standard protocol was replaced with 0.5 mM selenomethionine (final concentration).
Results
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The new RTS 500 E. coli HY Kit is based on the CECF
technology. As with the RTS 500 E. coli Circular Template
Kit, the device at the center of this technology includes
a 1-ml reaction chamber and 10-ml feeding chamber
separated by a 10 -kD membrane. This set-up allows
continuous replenishment of the critical components of
the transcription/translation reaction (Figure 1). Protein
expression proceeds for up to 24 hours, leading to
substantially higher yields compared to methods that do
not employ the CECF technology.
Several changes in the biochemistry have led to a significant increase in yield making the high-yield version up
to 10 times more productive than the RTS 500 E. coli
Circular Template Kit (Figure 2).
Because similar reaction conditions are also used in the
RTS 100 E. coli HY Kit, the two platforms can be used
together. The scalability allows economical screening of
conditions (e.g., different sequence variations for the same
gene) in the RTS 100 E. coli HY Kit with the possibility of
obtaining enough protein for extended analysis using the
same conditions in 24 hours with the RTS 500 E. coli HY Kit.
Since the protein yield is sufficient for structural analysis,
the incorporation of seleno-methionine was tested. Using
the same concentration of seleno-methionine instead of
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RTS
quantified via Coomassie-stained SDS-polyacrylamide
gels and – if required – via an activity test (e.g., fluorescence for GFP) using isolated protein as a reference.
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" Figure 3: Mass spectroscopy analysis of seleno-methionine-labeled
GFP. The shift in mass of 232 Da indicates the expected
incorporation of 5 seleno-methionine residues (5x46.9 = 234.5)
(Data kindly provided by N. Budisa and R. Huber, MPI for Biochemistry,
Martinsried/Germany)
methionine, all methionines could be readily replaced.
This was verified by mass spectroscopy (Figure 3).
Methionine is now separated from all other reagents in
the kit and can therefore be easily replaced.
Similarly, the exchange of amino acids for labeled amino
acids to obtain protein for use in NMR spectroscopy was
tested (see article by Ho S. Cho on page 27). For this purpose, all amino acids from the kit can be replaced with a
tailored amino acid mixture, using the RTS Amino Acid
Sampler and labeled amino acids of your choice.
Product
Pack Size
Cat. No.
RTS 500 Instrument
1 instrument
3 064 859
RTS 100 E. coli HY Kit
1 kit (24 reactions)
1 kit (96 reactions)
3 186 148
3 186 156
RTS 500 E. coli HY Kit
1 kit (2 reactions)
1 kit (5 reactions)
3 246 817
3 246 949
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