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ExpresswayTM Systems (Expresswayâ„? Plus, Expresswayâ„? Linear, Expresswayâ„? HTP, Expresswayâ„?
Tag-On-Demand)
TABLE OF CONTENT
PRODUCT DESCRIPTION
SHIPPING CONDITIONS
STORAGE CONDITIONS
STABILITY
QC SPECIFICATIONS
PROTOCOL & APPLICATION NOTES
Applications
Using non-E coli systems
Protocol overview
Expresswayâ„? Plus Scale-up of the Protein Synthesis Reaction
Specific component notes
Important considerations for the vector design
Expresswayâ„? linear TOPO elements
S30 extract
IVPS Buffer
Thermomixer
L-[35S] Methionine
Protein labeling
T7 RNA polymerase
RNase
DNase activity in the lysate
Expression resulting in a ladder of small sized products
Expression of specific proteins
Expression of membrane proteins
Expression using Champion pET vectors
Folding (Disulfide bond formation)
Glycosylation
Unusual amino acids
Purification of proteins produced from the Expresswayâ„? system
Use of protease inhibitors
Immunoprecipitation of proteins
Expresswayâ„? Lumio specific questions
ALTERNATE PRODUCTS & COMPATIBILITY
PRODUCT DOCUMENTATION
REFERENCES
PRODUCT NAME & CATALOG NUMBERS
COMPONENTS
ASSOCIATED PRODUCTS
1
PRODUCT DESCRIPTION
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The Expresswayâ„? Plus Expression System utilizes an efficient, coupled transcription and translation reaction to produce active
recombinant protein. The Expresswayâ„? Plus System eliminates the time-consuming steps of cell-based protein production such as
transformation, cell culture maintenance, and expression optimization steps. In just two hours, you can produce protein suitable for
an array of downstream applications and functional studies.
How It Works
The Expresswayâ„? Plus System provides all the components you need for optimal cell-free protein production. The kit includes an
E.coli extract containing the cellular machinery required to drive transcription and translation. The IVPS Plus reaction buffer
included in the kit contains the required amino acids (except methionine) and an ATP regenerating system for energy. The reaction
buffer is simply mixed with methionine, T7 Enzyme Mix, DNA template (driven by a T7 promoter) and E.coli extract. The newly
transcribed 5� end of the mRNA is bound by ribosomes and undergoes translation even as transcription of the remaining template
continues.
Some disadvantages of in vivo expression that can be overcome with the Expresswayâ„? Kit:
Toxicity to the host cell from over-expressed product
Product insolubility and formation of inclusion bodies
Rapid proteolytic degradation of the expressed protein
Incorporation of unnatural or modified amino acids
Incorporation of fluorescent probes into the protein
Requirement of high throughput analysis of protein products
Advantages over the original Expresswayâ„? Kit:
Optimization of the lysate and buffer result in:
More full-length/active protein produced
Improved yield
Greater flexibility
New Kit Composition
Modified IVPS reaction buffer- Methionine was removed from the amino acid mix and provided in a separate tube
allowing the researcher to add the necessary amount required for labeling
No slurry
Modified T7 RNA Polymerase Enzyme Mix
Modified S30 extract
The Expresswayâ„? Plus Linear Kit is optimized for the generation of active protein in a 2-hour reaction from a linear template
(rather than a plasmid). The kit contains an E. coli lysate that has been optimized for the production of increased levels of active
protein from linear templates when compared to the E. coli lysate included in the current Expresswayâ„? Plus kit.
Benefits of the Expresswayâ„? HTP Cell-Free E. coli Expression Kit
Convenience - just add circular or linear DNA template (5-20ng/ml final concentration) to the pre-mixed Expresswayâ„?
HTP mix
Flexible � Pre-mix in bulk format allows one to perform reactions in the plate format of one’s choice, e.g. 48, 96, 384-
well, clear or opaque material, round or flat bottomed
Fast � produce full-length, active protein in just two hours
Information on the web:
Beckman Biomek® FX Example method � A method file for a Biomek® FX equipped with an Span-8 head that
dispenses the Expresswayâ„? Reaction Mix into a 96-well plate, and a 96-channel head with disposable tips that can
add the DNA from a second plate
Import Files � Contains Labware definitions, Liquid Type definitions, and Techniques specific to Expressway�
HTP Expression
Tecan Freedom Example Script � A sample script for a Tecan Freedom using all default settings
Benefits of Expresswayâ„? Tag-On-Demand Cell-Free E. coli Expression System with Lumioâ„? Technology - A fast method
that uses a single DNA expression construct to express native or C-terminally tagged proteins
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It provides an in vitro platform for expressing the Ultimateâ„? Human ORF collection with C-terminal tags
It relies on the same DNA construct to express either tagged or native proteins
Full-length proteins are detected using the C-terminal Lumioâ„? Tag
The method uses a natural suppressor tRNA to discretionally add epitope tags onto a protein of interest
The efficiency of suppression is enhanced by the inactivation of the release factor 1.
A serine residue is added right after the last amino acid of the native protein and protein translation (elongation) continues
until reaching the first non-amber stop codon.
SHIPPING CONDITIONS
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All components are shipped on dry ice. Vectors may be shipped at room temperature.
STORAGE CONDITIONS
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Store all components at �800C.
The following components may be stored at �200C:
75 mM Methionine
DNase/RNase-Free Distilled Water
RNase A
T7 Enzyme Mix (after initial use only)
pT7/CAT Control Plasmid
pEXP1-DEST
pEXP2-DEST
pEXP3-DEST
pEXP1-GW/lacZ Control Plasmid
pEXP2-GW/lacZ Control Plasmid
Lumioâ„? Green Detection Reagent Proprietary
Lumioâ„? Gel Sample Buffer (4X)
Lumioâ„? In-Gel Detection Enhancer
pCR®2.1/T7-GFP Control Plasmid
T7 Promoter Primer #3
T7 Term Reverse Primer
2 ml Screw-Cap Tubes and lyophilized vectors may be stored at room-temperature.
STABILITY
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IVPS Plus E. coli Extract must be stored at �800C only.
2.5X IVPS Plus E. coli Reaction Buffer should be stored at -800C, but is stable at -200C as well.
Expresswayâ„? HTP Cell-Free E. coli Expression Mix should be thawed on ice and may be thawed up to 2 times without
loss of activity.
QC SPECIFICATIONS
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Each lot of the Expresswayâ„? Plus Expression System is functionally tested for protein generation by incorporation of 35S-
methionine. A 50 µl Expressway� Plus protein synthesis reaction must yield greater than 18 µg of CAT protein from the
pT7/CAT control plasmid.
PROTOCOL AND APPLICATION NOTES
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Applications
Using non-E coli systems
3
Protocol overview
Expresswayâ„? Plus Scale-up of the Protein Synthesis Reaction
Specific component notes
Important considerations for the vector design
Expresswayâ„? linear TOPO elements
S30 extract
IVPS Buffer
Thermomixer
L-[35S] Methionine
Protein labeling
T7 RNA polymerase
RNase
DNase activity in the lysate
Expression resulting in a ladder of small sized products
Expression of specific proteins
Expression of membrane proteins
Expression using Champion pET vectors
Folding (Disulfide bond formation)
Glycosylation
Unusual amino acids
Purification of proteins produced from the Expresswayâ„? system
Use of protease inhibitors
Immunoprecipitation of proteins
Expresswayâ„? Lumio specific questions
Applications
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Rapid scanning saturation mutagenesis
In vitro evolution methodologies such as ribosome display
Translation-based screening of mutagenized for shuffled genes and proteins
Drug screening (affecting translation rates)
Molecular diagnostic procedures such as the protein truncation test
Structure and function analysis of newly discovered gene product
In vitro protein engineering
Protein-protein interactions
Protein-RNA/DNA interactions
Post-translational modifications
Verification and characterization of products derived from cloned genes
Expresswayâ„? Linear is optimal for expression screening and high-throughput analysis
Using non-E coli systems
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Rabbit Reticulocyte Lysate (RRL) or Wheat Germ Extract (WGE) can often be used interchangeably for in vitro translation. In
general, RRL efficiently translates proteins greater than 30 kDa. WGE works well for proteins of all sizes (including those in the
range of 15-30 kDa), although larger proteins may be expressed more efficiently in RRL. This appears to be partially due to the
relative instability of small proteins in RRL. However, certain templates are translated more efficiently in RRL, whereas others
produce more protein in WGE; the difference in yield can be quite dramatic and must be determined empirically. Another
consideration is whether the protein of interest is present endogenously in either RRL or WGE, or whether factors that might
interfere with downstream applications may be present in RRL or WGE. This can be determined by performing a control in vitro
translation reaction with no template added. The RRL is recommended if Canine Pancreatic Microsomal Membranes are used for
co-translational processing (core glycosylation or signal peptide cleavage) or for production of proteins that require membrane
insertion for in vitro translation.
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Protocol overview and additional notes for each step
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Generate DNA template (PCR or plasmid vector)
Linear constructs work well with the Expresswayâ„? system if:
They contain the right promoter + spacer+ Ribosome Binding Site (RBS) + spacer + ATG and terminator sequences
Purified PCR products are used and 2-3 µg purified DNA for expression. pEXP vectors contain both promoter and
terminator sequences.
Purify template (commercial kits recommended)
Perform synthesis reaction
The extract and other components (T7 polymerase and buffer) can be premixed. In the current kit the researcher must
make a cocktail of the buffer, polymerase, and DNase/RNase free water and add it along with the template to the
extract.
2 hrs is standard but may go to 4 hours. The yield is protein dependent. For optimal yield, check expression at 1hr,
2hr, 3hr and 4hr. If protein is sensitive to protease, longer incubation is not recommended.
In a test that scaled up the reaction by 4-fold, to 200-250 µl, it was found that protein expression remained linear. A 2
ml tube is fine to use for scaled up reaction.
Any standard incubator or shaker can be used. Water bath gives better results than airflow incubator.
Analyze sample via Coomassie staining, western blot, bioactivity etc.
Coomassie stained gels will exhibit multiple bands coming from lysate.
The relative amount of full-length product is very important. In most cases an antibody that recognizes the C-terminal
part of the protein will detect mainly the full-length product whereas an antibody raised against the N-terminal will
detect many other isoforms (full-length and truncated proteins).
The positive control should be included. A lacZ assay or western blot should be performed as the band may not be easy to
visualize by Coomassie staining.
Expresswayâ„? Plus Scale-up of the Protein Synthesis Reaction (10ml volume)
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Linear scale up or down is ok. If large quantities of proteins is desired, Invitrogen has a customs service available.
I. Protein Synthesis
For each sample, add the following reagents to a 50 ml conical tube on ice.
Reaction Mix:
IVPS Plus E. coli Extract 4ml
2.5X IVPS Plus E. coli Reaction Buffer 4ml
T7 Enzyme Mix 200 µl
75 mM Methionine 200 µl
Total Volume 8.4 ml
Note: For trace labeling of the synthesized product, add 10 µl 35S Methionine to the reaction mix. Include a negative control (no
DNA) to determine background.
Add the following amount of DNA template to the reaction mix.
For plasmid DNA templates, add 100 µg
For linear DNA templates or PCR products, add 200µg
Bring the final reaction volume to 10ml with DNase/RNase-free water.
Gently vortex for 3 seconds to mix.
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Incubate tubes at 37°C for 2 hours in a standard shaking incubator (275-325 rpm) or in a water bath.
Note: It is possible to incubate tubes for up to 4 hours to obtain greater protein yield. In case of less soluble proteins,
incubate tubes at temperatures as low as 25°C to decrease the rate of protein synthesis and to promote proper folding. If
incubating tubes at temperatures lower than 37°C, it is recommended to extend the incubation time to 4 hours.
II. Product Analysis
For analyzing the 10 ml samples, take a 50 µl aliquot of the reaction and add 5 µl of RNase A, vortex briefly.
Place tubes in a microcentrifuge and briefly centrifuge.
Incubate samples in a thermo mixer (1,400 rpm) or water bath at 37°C for 15 minutes.
Perform TCA precipitations or SDS gel analysis according to the Expresswayâ„? Plus manual.
Specific component notes
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Important considerations for the vector design
Expresswayâ„? linear TOPO elements
S30 extract
IVPS Buffer
Thermomixer
L-[35S] Methionine
Protein labeling
T7 RNA polymerase
RNase
DNase activity in the lysate
Important considerations for the vector design
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T7 Promoter: Transcription of the gene of interest must be driven by the T7 promoter (not the T7lac promoter).
Some of the traditional pET vectors contain the T7lac promoter and the lacI gene, which render poor yields. This is
because the lac repressor (encoded by the lacI gene) binds and represses transcription from this promoter. The lac
repressor should not interfere with the expression from a regular T7 promoter and no IPTG should be needed in that
case.
There are no data for the behavior of vectors other than pEXP1, pEXP2, and pCRT7 (N- and CT). It is advisable to
pre-determine that the components in the vector (T7 Promoter, RBS, ATG, and stop site) are appropriately
configured. For questions regarding the use of vectors or elements for optimal expression, please see Expressions 9.3,
page 5.
T7 terminator: The T7 terminator is important for efficient in vitro transcription from a supercoiled plasmid.
Pyrophosphate released from RNA synthesis will bind Mg++ to form insoluble MgPO4. If the T7 terminator is
absent, long, nonspecific RNA products will be produced which can deprive the reaction of dNTPs and generate
copious amounts of pyrophosphate.
Gene 10 Sequence: A gene10 sequence enhances the stability of the in vitro expressed sequence. It is thought that
this sequence causes a specific stem-loop structure to form, which helps to stabilize the mRNA, and leads to
increased translation. The gene 10 sequence, part of the ribosome binding site (RBS), is part of the pEXP-DEST and
pCRT7 vectors, even though this is not shown as part of the vector map.
Mini cistron: The mini cistron (as in the Trc vectors) also acts to enhance translation by coding for a little gene
sequence that makes a small peptide. Since this brings the translation machinery in the proximity of the start of the
gene of interest, it gets the system just downstream going.
Copy number: It is recommended to start out with a high copy number plasmid. This way, minipreps (e.g. SNAP)
could be used directly with Expresswayâ„? . Many of the pET vectors are low copy, and need to be concentrated
before being used in an in vitro expression system.
Spacing: spacing between RBS and ATG is very important for efficient translation. Check the spacing and presence
of T7 terminator to determine compatibility with Expresswayâ„? .
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RBS: RBS will increase the yield of protein and increase the translation fidelity.
Expresswayâ„? linear TOPO elements
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The reading frame setup of the PCR product: If you want to use the V5-His tag from the 3' TOPO element, make sure the last three
bases before the 11 base addition is a functional codon; if you want your gene of interest in the PCR product to be in frame with
the start codon from the 5' TOPO element, put the first functional codon immediately after the 11 base addition.
S30 extract
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Adding RNase inhibitor to the reaction may increase protein yield.
It is very important to store the extract at �800C
The extract is made from an E. coli containing no recombinant plasmid, gene or protein . The pH of the buffer is 7.5.
There is no EDTA, EGTA or other chelating agent present in the IVPS mix. However addition of 0.5mM EGTA should
not affect the extract activity.
Addition of purified ribosomes to the extract does not increase translation activity. The concentration of active
ribosomes in the extract is high .
No detergents or lipids are added to the Expresswayâ„? Plus kit. However, they can be added by the researcher to help
with solubility of proteins.
The cell-free extract provided with the kit contains ribosomes that are not spectinomycin resistant and should be
inactivated after the addition of the antibiotic. You may use the system to test other ribosomes in the presence of
spectinomycin, provided that it doesn’t have its own translation factor (are not active in translation).
Expresswayâ„? does not contain any calcium
There is no glucose or lactose in the Expresswayâ„? system. Medium used for growing E. coli cells is 2X YT without
glucose or lactose. Also, no glucose or lactose is added to E. coli cell-free extract.
There has been no knowledge of esterase activity in E. coli or E. coli extract
The E. coli strain in use has very little suppressor activity, and it is not a Sup F or Sup E strain. Therefore, it should be
useful for introducing modified amino acid(s).
Some of the enzymes involved in amino acid metabolism are present and active in the extract, but don't have any specifics
about which ones in which amounts. The only one known for sure is that glutamate is slowly degraded, even in the
absence of protein synthesis (perhaps converted to other amino acids). There have been literature reports regarding other
cell-free extracts where cysteine, serine and threonine are also rapidly degraded, but again there has been no examination
in the extract.
Asparaginase may be present (at low levels), but it has not been looked for it specifically. Testing heavy isotope
asparagine incorporation by mass spec has been done., and it was found >90% incorporation of the 15N asparagine, so it
is unlikely that any Asparagine is getting degraded as it is incorporated very efficiently. Synthesized asparaginase should
be present in much higher levels than any that is present in the extract itself. One should be a little concerned that perhaps
the synthesized asparaginase may degrade the available pool in the reaction and shut down synthesis of the enzyme itself
(since it also contains asparagine), so they may want to supplement the reaction with more asparagine if this is the case.
DNase treatments of the extracts are not done, but almost no expression of any endogenous proteins in the cell-free
reaction is seen (each lot of extract is tested to ensure no/low background expression).
E. coli contain some chaperone proteins used for protein folding.
The buffer used is HEPES.
The Expresswayâ„? system is not tested for endotoxins or LPS (lipopolysaccharides).
IVPS Buffer
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IVPS buffer contains DTT that may interfere with binding of His-tags to a nickel column. However, after 2 hours
incubation at pH 8.0, most of the DTT should be oxidized and in such form should have no negative effect on binding.
Unincorporated histidine may also "compete" for binding to Ni, but this problem can be solved by using a bigger column
to increase capacity.
The reaction mix is quite complex, and is the main source for amino acids. There are some endogenous amino acids that
are also present in the extract, and while many of these will not be complexed with tRNAs, it can not be guaranteed that
even if an amino acid were left out of the reaction mix, that there would not be activated amino acids in the extract.
Thermomixer
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The Eppendorf Thermomixer can be bought from Fisher (Fisher catalog # 05-400-200). It costs ~ $1700. This is a
versatile incubator and can be used as a general incubator, as well as a shaking incubator. It can hold 24 samples at one
time and the temperature is adjustable from 4°C to 96°C. A Thermomixer that can accommodate both plates and tubes is
available that can shake as high as the 1400 rpm that is needed for best results with Expresswayâ„? .
Using a vortexer at 37 °C has been tested and it is not recommended. It is believed this particular method heats the
samples above 37 °C and greatly decreases the yields.
If a researcher does not want to use the Thermomixer, they can use an incubator, water bath or water bath with shaker.
Both 1.5ml and 2ml tube can be used. Water bath, water bath with shaker or airflow incubator gives compatible protein
yield as in the Thermomixer.
L-[35S]Methionine
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Supplier: Amersham Biosciences
Product Pack Size Code Number
L-[35S]Methionine 18.5 MBq, 500 µCi SJ204-500µCi
37 MBq, 1 mCi SJ204-1mCi
74 MBq, 2 mCi SJ204-2mCi
185 MBq, 5 mCi SJ204-5mCi
20mM potassium acetate solution containing 0.1% 2-mercaptoethanol.
> 37 TBq/mmol, > 1000 Ci/mmol.
555 MBq/ml, 15 mCi/ml.
CH3S(CH2)2CH(NH2)CO2H
Protein labeling
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Extracts without Methionine:Currently, this is not offered.
35
S labeling when 35S labeled amino acid is added will give very specific labeling (>98%) of newly synthesized protein
coming off mRNA made by T7 RNA polymerase. There may be some minor non-specific labeling (0-2%).
Labeling using Leucine: To label with leucine rather than methionine, the final concentration of leucine in the 50-µl
reaction should be approximately 1.25 mM.
Shorter bands on the gel could be due to:
Incomplete peptide chains
Ribosomal pausing
Presence of rare codons and tRNAs
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Lack of amino acids
RNA degradation
DNA degradation
Protein degradation
T7 RNA polymerase
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If researchers run out of this enzyme, they can substitute with the standalone T7 RNA polymerase (50unit/µl, cat no.18033019).
Use 1-1.5ul in 50ul reaction system.
RNase
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The only purpose for the RNase A treatment is for researchers that are radiolabeling their protein and wish to quantitate yields by
TCA precipitation. In this method, trichloroacetic acid is added to the reaction, which causes all larger molecules to precipitate.
This solution is then passed over a glass fiber filter which collects the larger molecules, including the synthesized protein, a
majority of proteins from the extract and charged tRNAs, The smaller molecules like the unincorporated radiolabel pass through.
The radioactivity collected on the filter is then quantitated in a scintillation counter. If the treatment is omitted, the 35S Met-
charged tRNA will also collect on the filter, thus generating additional counts not from the synthesized protein.
Researchers may also add 1M NaOH instead of RNase A. The NaOH hydrolyzes the aminoacyl-tRNA bond effectively removing
the radiolabel from the tRNA, and allowing it to pass through the membrane.
If a researcher is not planning on measuring radiolabel incorporation by TCA precipitation, they need not add the RNase A at all.
You can proceed to SDS-PAGE analysis or activity assays directly.
DNase activity in the lysate
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If there is DNA-protein interaction between the protein produced and the DNA template, DNase activity will be a concern. DNase
activity exists in every in vitro expression system. The advantage of the Expresswayâ„? system is that it is by far the most robust
system for in vitro protein production. If you are using systems from other companies, you may need 1.5 to 2 hours to produce
sufficient protein. But with Expresswayâ„? , you will obtain considerable amount of protein within 30 minutes. DNA degradation
occurs relatively slowly. Within 30 minutes, there is a good chance of recovering a majority of the DNA template. It is suggested
to perform a time course experiment at 30, 60, 90 and 120 minutes and choosing the best time point for obtaining a good yield of
protein and recovering most of the DNA template at the same time. Plasmid DNA is more stable than linear DNA in the in vitro
expression system. If a plasmid construct can be used as a template, there is a better chance to go for 2 hours and still recover the
majority of the template DNA.
Expression resulting in a ladder of small sized products
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There may be many reasons for such an outcome. The most common are: proteolysis, degradation of DNA and/or RNA templates
(truncated templates will generate truncated protein products), internal initiation (if there are many methionines and internal RBS-
like sequences in the gene, ribosome may initiate translation from the wrong methionine), premature termination, translational
pausing, frequent rare codon usage, complicated secondary structure of RNA and others.
9
Often shorter products are still attached to ribosomes as peptidyl-tRNA complex and can be removed from the reaction by
ultracentrifugation (Beckman analytical ultracentrifuge). The material remaining in the supernatant mostly corresponds to full-
length products. Also high-speed centrifugation (10 min at 10,000Xg) may remove aggregated material and help to lower the
background.
Expression of specific proteins
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Expression level (µg/50 µl reaction)
Protein Size (kDa)
CAT 24 19.1
Sigma 32.2 11.66
GFP 39.4 18.0
t-Tak 44 11.85
Vraf 46.4 38.55
TK 47.7 20.7
Bk2 48 25.2
3aK 62 30.62
DB1 64 26.78
GUS 75.6 45.28
Bgal 127 36.65
Bgal, GFP, GUS and CAT were all tested for activity following expression in Expresswayâ„? , and found to be functional.
Expresswayâ„? Linear System- approximate yields for specific proteins
CAT- approximately 9 µg/50 µl reaction
GFP- approximately 10 µg/50 µl reaction
GUS- approximately 8 µg/50 µl reaction
LacZ approximately 8 µg/50 µl reaction
.
Expression of membrane proteins
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Membrane proteins are highly hydrophobic and present solubility problems usually requiring phospholipids or detergents to mimic
the natural environment. The following may be added to reaction mixes to try to increase solubility: The indicated concentrations
are final concentrations in the reaction mix.
Triton X-100 (0.5 - 1%),
Dodecyl-maltoside (0.025 - 0.05%), CHAPS (0.1 - 2%),
Octylglucoside (1 - 2mM), Brij 35 (0.5 - 0.1%),
Octyl-gluco-pyranoside (1 - 1.5%).
Membrane proteins require special precautious. They are proteolytically sensitive and may require protein-folding catalysts for
proper folding.
LacZ control: β-Gal is a large protein and has a tendency to aggregate. in vitro synthesized β-Gal is highly active. Efficiency of
binding of β-Gal to Ni-NTA may increase under denaturing conditions but the purified protein will be inactive. The activity of β-
Gal may be rebuilt after re-folding. Invitrogen's ProBond Purification kit has a hybrid purification protocol, which initially
denatures the protein but elutes in a native buffer to partially restore activity (depending on the protein). The pEXP2-lac Z control
has a tendency to aggregate less than pEXP1-Lac Z. Depending on the goals (yield or activity) the researcher will need to decide
what purification method to use. The LacZ control contains 27 Methionies and an apparent MW of 128 kDa.
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Addition of some detergents (i.e. mM CHAPS, 0.2 mM Triton X-100, 1.5 mM Brij 35) at the beginning of transcription/translation
reaction may positively affect solubility of synthesized proteins. CHAPS, n-Dodecylmaltoside or Triton X-100 added at 0.1% -1%
concentration added at the beginning can increase solubility.
Lowering the amount of T7 RNA polymerase by 1/2 or 1/4 may be needed. Large amounts of synthesis could overwhelm the
folding machinery.
Temperature: Incubation at 30oC gives more soluble proteins in comparison to 37oC. Researchers can go down to 25oC (at 25oC,
do the reaction for 4 hrs instead of 2 hrs).
Expression using Champion pET vectors
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Several of the pET vectors with Expresswayâ„? have been tested and they work fine. Expression levels can vary significantly just
by moving theposition of the tag (C- vs. N- terminal).
Folding (Disulfide bond formation)
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It is known that disulfide bridges will not form in this system. If a researcher would like disulfide bridges to be formed, the use of
Iodoacetamide is recommended (Biotechnol Bioeng. 2004 Apr 20;86(2):188-95 PMID: 15052638). Pre-incubation of the extract
with 3 mM Iodoacetamide for 30 min at room temperature is recommended. The reaction already contains 1 mM DTT (= 2mM
sulfhydryls), therefore only 1 mM Iodoacetamide will be in excess. Another approach would be using oxidized glutathione or
oxidized DTT (use at least 2-3 mM). Please be advised that this may compromise the yield considerably.
Glycosylation
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The machinery for glycosylation is absent in these extracts.
Unusual amino acids
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A protocol for adding radiolabeled amino acids is provided in the manual.
Purification of proteins produced from the Expresswayâ„? system
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Suggested protocol:
The capacity of most Ni-NTA columns is 20 - 50 mg per 1ml of the beads. In any case, the researcher will need to know the
capacity of the resin. Assuming, that the protein of interest is synthesized at unusually high levels (e.g. 50 µg protein per 50 µl
reaction), the researcher may need to use just 1 µl of resin to bind all the synthesized protein (50 mg protein/1 ml of beads or 50 µg
protein/1ul). In such a case, the researcher should add 5 µl of resin to the 50 µl reaction and purify the product on a batch-mode
(not a column). After binding for 2 - 5 min, spin the resin down for 1 min using a low-speed centrifugation (bench type), wash the
resin with binding buffer to eliminate contaminants, spin the resin down again then elute the protein with a small amount (10-20
µl) of eluting buffer and separate the unbound resin' by centrifugation. Save the last supernatant as a source of the synthesized
protein.
Use of protease inhibitors
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(back to Protocol and Application Notes)
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If needed, it is recommended the addition of PMSF (final conc. 0.5-1.0 mM) at the beginning of the Expresswayâ„? reaction. It is
better to dissolve PMSF in isopropanol instead of EtOH. EtOH has a negative effect on protein synthesis. The other useful protease
inhibitor is Pafablock C (AEBSF, Sigma). It has a longer half-life compared to PMSF (2 h compared to 30 min). Use 0.1- 0.2 mM
AEBSF in the transcription/translation reaction. Both PMSF and Pafablock C are serine protease inhibitors.
Immunoprecipitation of proteins from an Expresswayâ„? reaction.
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(back to Protocol and Application Notes)
Immunoprecipitation of proteins from the Expresswayâ„? reaction has not been tested yet.
Expresswayâ„? Lumio specific questions
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(back to Protocol and Application Notes)
Measuring 1 picomole of the Lumioâ„? fusion protein spectrophotometrically:
It is not possible to measure the concentration by spectrometry since there are copious amounts of proteins in the E. coli
extract. The lower detection limit for in-gel detection using the Lumio Reagent is 1pmol.
Stopping point for this procedure:
The reactions may be frozen directly after incubation, or after aliquots are taken for acetone precipitation, or after
resuspending in sample buffer. However, once the Lumio detection reagent is added, the samples must preferably be used
immediately. After resuspending the sample in loading buffer containing the Lumio reagent, the remaining sample may
be frozen and rerun on the gel if the researcher adds more enhancer after heat denaturation and before loading.
Gel recovery from photobleaching if overexposed:
The signal should recover, depending on how long the gel was overexposed. Photobleaching doesn't happen that quickly
though. The researcher should get the image within 15-20 minutes. It is more a question of sensitivity: The more the
reagent is exposed, the lower the signal becomes.
Storing the gel before adding enhancer solution:
The gel cannot be stored. The samples may be stored in the Lumio Gel Sample Buffer before adding the Detection
Reagent or the enhancer.
PRODUCT DOCUMENTATION
(back to Table of Content)
Brochures Citations COA
FAQ Licensing Manuals
MSDS Vector Data
REFERENCES
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Focus 25.2, page 28-32.
Note: There is an error on page 30. The figure 2 legend part A is not the correct information for this figure.
Focus 25.3, page 3-6.
Expressions 9.3, page 5.
PRODUCT NAME AND CATALOG NUMBERS
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Name Size Catalog Number
ExpresswayTM Plus Expression System 20 reactions K9900-10, K9900-20 (with pEXP1-
DEST) K9900-30 (with pEXP2-DEST)
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DEST), K9900-30 (with pEXP2-DEST),
V960-01, V960-02 [K990010,K990020,
K990030, V96001, V96002]
Expresswayâ„? Plus Expression System with Lumio 20 reactions K9900-60, K9900-70, V960-03
Technology [K990060, K990070, V96003]
Expresswayâ„? Linear Expression System 20 reactions K9900-40 [K990040]
Expresswayâ„? Linear Expression System with TOPO Tools 20 reactions K9900-50, T310-20, T312-20 [K990050,
Technology T31020, T31220]
Expresswayâ„? HTP Cell-Free E. coli Expression Kit 20 reactions K9900-80 [K990080]
Expresswayâ„? Tag-On-Demand Cell-Free E. coli Expression 20 reactions K9900-90, K9900-95, V960-04
System with Lumio Technology [K990090, K990095, V96004]
pEXP1-DEST Vector Kit (includes positive control vector, V96001
6µg
pEXP1-GW/lacZ, 10ug)
6 µg
pEXP2-DEST Vector Kit (includes positive control vector, V96002
pEXP2-GW/lacZ, 10ug)
6 µg
pEXP3-DEST Vector Kit (includes positive control vector, V96003
pEXP3-GW/CAT, 10ug)
6 µg
pEXP4-DEST Vector Kit (includes positive control vector, V96004
pEX4-ORF, 10ug)
COMPONENTS
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Name Size Part Number Catalog Number
Expresswayâ„? Plus Expression System (IVPS Plus E. coli Extract, 20 reactions 451004 K9900-10,
2.5X IVPS Plus E. coli Reaction Buffer, 75 mM Methionine, K9900-20,
DNase/RNase-Free Distilled Water, RNase A, T7 Enzyme Mix, K9900-30
pT7/CAT Control Plasmid, 2 ml Screw-Cap Tubes)
6 µg (10 µg
pEXP1-DEST Gateway Vector Kit (pEXP1-DEST, pEXP1- 430112 K9900-20,
GW/lacZ) V960-01
control)
6 µg (10 µg
pEXP2-DEST Gateway Vector Kit (pEXP2-DEST, pEXP2- 430113 K9900-30,
GW/lacZ) V960-02
control)
Expresswayâ„? â„? Plus Expression System with Lumioâ„? 20 reactions 451006 K9900-60,
Technology module (IVPS Lumioâ„? E. coli Extract, 2.5X IVPS K9900-70
Plus E. coli Reaction Buffer, 75 mM Methionine,
DNase/RNase-Free Distilled Water, T7 Enzyme Mix, pEXP3-
GW/CAT Control Plasmid, 2-ml Screw-Cap Tubes)
Lumioâ„? Green Detection Kit module (Lumioâ„? Green
Detection Reagent, Lumioâ„? Gel Sample Buffer, Lumioâ„? In-
Gel Detection Enhancer) LC6090
6 µg (10ug
pEXP3-DEST and pEXP3-GW/CAT 433026 K9900-70,
V960-03
control)
Benchmark� Fluorescent Protein 125 µl LC5928 K9900-70
Standard
Expresswayâ„? HTP Cell-Free E. coli Expression Mix 5 x 2.5 ml 451010 K9900-80
(Expresswayâ„? E. coli Extract, Expresswayâ„? 2.5X E. coli
Reaction Buffer containing methionine, T7 Enzyme Mix, pT7/CAT
Control Plasmid)
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Expresswayâ„? Linear Protein Synthesis Reagents (Box 1) (IVPS 451005 K9900-40
Ultra E. coli Extract, 2.5X IVPS Plus E. coli Reaction Buffer, 75
mM Methionine, DNase/RNase-Free Distilled Water, RNase A, T7
Enzyme Mix, 2 ml Screw-Cap Tubes)
And
Expresswayâ„? Linear Control Reagents (Box 2) (pCR2.1/T7-GFP 451007
Control Plasmid, T7 Promoter Primer #3, T7 Term Reverse Primer)
Expresswayâ„? Linear Protein Synthesis Reagents (IVPS Ultra E. 451005 K9900-50
coli Extract
2.5X IVPS Plus E. coli Reaction Buffer, 75 mM Methionine,
DNase/RNase-Free Distilled Water, RNase A, T7 Enzyme Mix, 2 ml
Screw-Cap Tubes)
Expressway� Linear PT7 5´ Element 451008
Expressway� Linear V5-His T7 Term 3´ Element 451009
Expresswayâ„? Tag-On-Demandâ„? Protein Synthesis Reagents 452004 K9900-90
(Expresswayâ„? Tag-On-Demandâ„? slyD
Mutant E. coli Extract, Expresswayâ„? 2.5X E. coli Reaction Buffer,
Expresswayâ„? Tag-On-Demandâ„?
Suppressor tRNA, 75 mM Methionine, DNase/RNase-Free Distilled
Water, RNase A, T7 Enzyme Mix, 2 ml Screw-Cap Tubes)
pEXP4-DEST Vector 430517
Lumioâ„? Green Detection Kit LC6090
BenchMarkâ„? Fluorescent Protein Standard LC5928
ASSOCIATED PRODUCTS
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(Please consult the product manual to determine which of the following products would be relevant for your application)
DNase/RNase-Free Distilled Water, 500 ml, catalog # 10977-015
S.N.A.P. MiniPrep Kit, 25 reactions, catalog # K1900-01
Coomassie Brilliant Blue R-250 Protein Stain, 10 g, catalog # 15528-011
β-Gal Assay Kit, 100 reactions, catalog # K4155-01
β-Gal Antiserum, catalog # R901-25 (sufficient for 25 Western blots)
CAT Antiserum, catalog # R902-25 (sufficient for 25 Western blots)
Gateway LR Clonase Enzyme Mix, 20 reactions, catalog # 11791-019
Ampicillin, 20 ml (10 mg/ml), catalog # 11593-019
PureLinkâ„? HQ Mini Plasmid Purification Kit, 100 reactions, catalog # K2100-01
Library Efficiency DB3.1 Competent Cells, 1 ml (5 x 0.2 ml), catalog # 11782-018
Coomassie Brilliant Blue R-250 Protein Stain, 10 g, catalog # 15528-011
AcTEV Protease, 1,000 units, catalog # 12575-015
CAT Antiserum, catalog # R902-25 (sufficient for 25 Western blots)
BenchMark Fluorescent Protein Standard, 125 µl, catalog # LC5928
ProBond Purification System, 6 purifications, catalog # K850-01
E-PAGE 96 6% High-Throughput (HTP) Protein Electrophoresis System, 1 Starter Kit, catalog # EPST96-06
SimplyBlue SafeStain, 1 L, catalog # LC6060
Lumio Green Detection Kit, 1 kit, catalog # LC6090
Expresswayâ„? Linear Expression System with TOPO Tools Technology, 20 reactions, catalog # K9900-50
Expressway� Linear PT7 5΄ Element, 20 reactions, catalog # T310-20
Expressway� Linear V5-His T7 Term 3΄ Element, 20 reactions, catalog # T312-20
Platinum Taq DNA Polymerase High Fidelity, 100 units 11304-011
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GFP Antiserum, 50 µl, catalog # R970-01 (sufficient for 25 Western blots)
For detecting recombinant protein expression from pEXP1-DEST constructs, the following antibodies may be used:
Anti-Xpress Antibody, catalog # R910-25
Anti-Xpress-HRP Antibody, catalog # R911-25
Anti-HisG Antibody, catalog # R940-25
Anti-HisG-HRP Antibody, catalog # R941-25
Anti-HisG-AP Antibody, catalog # R942-25
For detecting recombinant protein expression from pEXP2-DEST constructs, the following antibodies may be used:
Anti-V5 Antibody, catalog # R960-25
Anti-V5-HRP Antibody, catalog # R961-25
Anti-V5-AP Antibody, catalog # R962-25
Anti-His(C-term) Antibody, catalog # R930-25
Anti- His(C-term)-HRP Antibody, catalog # R931-25
Anti- His(C-term)-AP Antibody, catalog # R932-25
For detecting recombinant protein expression from pEXP3-DEST constructs, the following antibodies may be used:
Anti-HisG Antibody, catalog # R940-25
Anti-HisG-HRP Antibody, catalog # R941-25
Anti-HisG-AP Antibody, catalog # R942-25
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