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View additional product information for Expressway™ Maxi Cell-Free E. coli Expression System - FAQs (K990097)
43 product FAQs found
Unfortunately, this may result in a loss of activity.
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We would recommend using T7 RNA polymerase (Cat. No. 18033019, 50 U/µL). Use 1-1.5 µL in a 50 µL reaction system.
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Smearing may occur if samples for the following reasons:
- Samples were not precipitated with acetone: precipitate proteins with acetone to remove background smearing.
- Too much protein was loaded: reduce the amount used.
- The gel itself was not clean: rinse the gel briefly before exposing to film.
- Ethanol was present in the protein synthesis reaction: make sure that any residual ethanol is removed during DNA purification.
- Check the date of your pre-cast gels: do not use gels after the expiration date.
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There may be several reasons for why this is occurring. 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, the ribosome may initiate translation from the wrong methionine), premature termination, translational pausing, frequent rare codon usage, complicated secondary structure of RNA, and others. This can also happen if proteins are denatured for too long, or not enough SDS was added to the 1X SDS-PAGE sample buffer.
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- Your protein may not be folding properly: try to reduce the incubation temperature to as low as 25 degrees C during synthesis.
- You may require post-translational modification of your protein: the Expressway system will not introduce post-translational modifications to the recombinant protein.
- Your synthetic protein may require co-factors for complete activity: try adding required co-factors to the protein synthesis reaction.
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If you are getting no protein from your control reaction, the reagents may have lost activity or may be contaminated with RNases. Check the storage conditions and expiration of the reagents. Use care when freezing and thawing the Expressway E. coli slyD-Extract, Expressway 2.5X IVPS E. coli Reaction Buffer, and Expressway 2X IVPS Feed Buffer. One or two freeze/thaw cycles are acceptable, but avoid multiple cycles.
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Protein yield may decrease as the size of the protein increases. You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis.
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You can try to reduce the incubation temperature to 25-30 degrees C during protein synthesis. Additionally, a mild detergent can be added (e.g., up to 0.05% Triton-X-100, 0.025% sodium dodecyl maltoside, 0.1% CHAPS, or 0.05% Brij-58) to the reaction and feed buffer. You can also try to add molecular chaperones to the reaction.
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Please review the following suggestions:
- Check the sequence of your vector (ATG initiation codon, in frame, etc.).
- If working with a N- or C-terminal tag, the tag may be affecting the RNA structure and lowering translation levels. Try moving the fusion tag or the other terminus.
- Ensure that your DNA template is pure, and not contaminated with ethanol, sodium salt, ammonium acetate, or RNases.
- Do not purify your DNA from an agarose gel, as this can inhibit the reaction.
- We recommend using 10-15 µg of template DNA in a 2 mL protein synthesis reaction. If you are expressing a large protein, increase the amount of DNA template used in the protein synthesis reaction to 20 µg.
- Ensure that you are using a thermomixer or incubator with shaking, as opposed to a non-shaking incubator or water bath for the reaction.
- Multiple feeding steps can further improve the protein yield. Instead of doing one feeding at 30 min of the initial reaction, you can feed the reaction multiple times with smaller volumes of feed buffer to the sample more frequently (i.e., 0.25 mL feed buffer to 1 mL sample every 45 min over 3 hours) after initiating protein synthesis.
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There are several ways to analyze your samples after the protein synthesis reaction, including: Coomassie-stained protein gel analysis, western blot analysis, enzymatic activity assay, or by affinity purification (if an affinity tag is present). If you plan to analyze your sample using polyacrylamide gel electrophoresis, you should first precipitate the proteins with acetone to remove background smearing. A protocol to perform acetone precipitation and other general guidelines for gel electrophoresis are provided in the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) on page 22.
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If needed, we recommend the addition of PMSF (final concentration 0.5-1.0 mM) at the beginning of the Expressway reaction. It is better to dissolve the PMSF in isopropanol instead of ethanol, as ethanol has a negative effect on protein synthesis. You can also use Pefabloc SC (final concentration 0.1-0.2 mM AEBSF) in your transcription/translation reaction. Both are serine protease inhibitors.
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No, unfortunately, the machinery for glycosylation is absent in these extracts.
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We have not specifically tested for this, although we do know that the limiting factor will be the tRNAs. Because of this, simply adding more of a particular amino acid will not make a difference.
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We have not actually done any purifications with the extracts using the His tag. However, it should work, especially if you do it under denaturing conditions.
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No. Disulfide bridges will not form in this system. However, the formation of disulfide bridges may be achieved through the addition of iodoacetamide (Biotechnol Bioeng 2004, 86(2):188-195). Pre-incubation of the extract with 3 mM iodoacetamide for 30 min at room temperature is recommended. The reaction already contains 1 mM DTT (equivalent to 2 mM sulfhydryls); therefore, only 1 mM iodoacetamide will be in excess.
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E. coli cells do indeed contain some chaperone proteins used for protein folding. However, extra chaperone proteins were not added to the extract.
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Methionine is supplied separately in the kit to allow you to incorporate unnatural amino acids into your recombinant protein and adjust the amino acid concentration in the protein synthesis reaction. Depending on your application, you may use the following unnatural amino acids:
- Radiolabeled methionine: Use 35S-methionine to produce radiolabeled protein for use in expression and purification studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled and unlabeled methionine.
- Heavy metal-labeled methionine: Use selenomethionine (Budisa et al., 1995 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2374119/]; Doublie, 1997 [http://www.ncbi.nlm.nih.gov/pubmed/9048379]; Hendrickson et al., 1990 [http://www.ncbi.nlm.nih.gov/pubmed/2184035?dopt=Abstract]) to produce labeled protein for use in X-ray crystallographic studies. See “Performing the Protein Synthesis Reaction” on page 21 of the manual (http://tools.thermofisher.com/content/sfs/manuals/expressway_system_man.pdf) for recommended amounts of labeled methionine. Note: When using selenomethionine, do not use any unlabeled methionine in the protein synthesis reaction.
When setting up the protein synthesis reaction:
- To generate radiolabeled protein using 35S-methionine, use 2 µL of 35S-methionine and 1 µL of unlabeled 75 mM methionine.
- To generate labeled protein using selenomethionine, use 2 µL of selenomethionine only; do not add unlabeled methionine.
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You may obtain your protein of interest in as little as 2.5 hours of incubation after feeding (3 hours total). Many reactions yield 80-90% of total protein within 3 hours. However, for maximum yield, we recommend incubating the reaction for a full 6 hours.
Additionally, higher protein yields may be obtained by adding one half-volume of feed buffer at 30 minutes and one half-volume of feed buffer again at 2 hours after initiating the protein synthesis reaction.
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We recommend incubating the protein synthesis reaction at a temperature range from 30-37 degrees C. The optimal temperature to use depends on the solubility of your recombinant protein, and should be determined empirically. Higher protein yields are generally obtained with incubation at higher temperatures (i.e., 37 degrees C); however, protein solubility generally improves with incubation at lower temperatures (i.e., 30 degrees C).
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To obtain optimal protein yield, it is critical to mix the reaction thoroughly throughout the incubation period. We recommend using a thermomixer incubator set to 1,200 rpm or a shaking incubator set to 300 rpm. Do not use stationary incubators such as incubator ovens or water baths, as protein yields may be reduced by up to 30-50%.
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For screening reactions, the standard volume is 100 µL (50 µL initial reaction + 50 µL feed buffer), but this can be decreased to 25 µL reaction volume and increased up to 2 mL reaction volume. Note that protein yields may vary depending on the nature of the protein expressed and the template used.
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The standard reaction time is 2 hours. However, increasing the time to 4 hours may increase the yield of protein. For less soluble proteins, this longer incubation should be carried out at ambient temperature.
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No, the proper machinery for glycosylation is not present in these extracts.
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Some Invitrogen pET vectors work well in this system; however, the yields might be lower than that found with other vectors due to the presence of the T7lac promoter. The lac repressor can bind to the lac operator site and interfere with expression even when IPTG has been added to the reaction. The best vectors to choose are the pEXP-DEST vectors, pEXP5-TOPO vectors, and pRSET vectors. In addition to the T7 promoter, these also have a gene sequence that enhances translatability.
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No, the E. coli strain is not a supF or supE strain, and it has very little suppressor activity. Therefore, it should be useful for introducing modified amino acids.
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Yes. We recommend starting out with a high copy number plasmid. This way, a researcher can go right from a miniprep kit directly into the Expressway reaction. Many of the pET vectors are low copy, and need to be concentrated before being used in an in vitro expression system.
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We have looked at two proteins: one that was totally insoluble in intact E. coli and another that was partly insoluble in E. coli. In both cases, we saw at least some soluble protein when synthesized in vitro with the Expressway system. In these instances, it did seem that there is at least some increased solubility with the Expressway system. However, synthesizing protein in vitro does not completely change protein solubility.
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No, we do not recommend doing so, as we have seen this inhibit the protein synthesis reaction. Instead, you can use commercial DNA purification kits (such as our PureLink HQ Mini Plasmid Purification Kit) or a CsCl gradient centrifugation to purify your DNA template.
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You can use supercoiled plasmid DNA, linear DNA, or a PCR product as your template. For proper expression, all templates must contain a T7 promoter, an initiation codon, and a prokaryotic Shine-Dalgarno ribosome-binding site (RBS) upstream of the gene of interest. If you are designing your own expression construct, we recommend generating a DNA template with the following elements:
- Gene of interest placed downstream of a T7 promoter and a ribosome-binding site (RBS). The gene of interest must contain an ATG initiation codon and a stop codon.
- Sequence upstream of the T7 promoter containing a minimum of 6-10 nucleotides (nt) for efficient promoter binding (required for linear PCR products). This sequence need not be specific.
- Sequence following the T7 promoter containing a minimum of 15-20 nt, which forms a potential stem-and-loop structure as described by Studier et al., 1990.
- Sequence of 7-9 nt between the RBS and the ATG initiation codon for optimal translation efficiency of the protein of interest. This sequence need not be specific.
- A T7 terminator located 4-100 nt downstream of the gene of interest for efficient transcription termination and message stability.
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- Transcription of the gene of interest must be driven by a T7 promoter (not the T7lac promoter). Using a T7lac promoter typically renders poor yield, as the lac repressor encoded by the lacI gene binds and represses transcription from this promoter.
- The T7 terminator is important for efficient in vitro transcription from a supercoiled plasmid. If the terminator is absent, long nonspecific RNA products will be produced, which can deprive the reaction of dNTPs and generate a copious amount of pyrophosphate.
- A gene10 sequence enhances the stability of the in vitro expressed sequence. This sequence causes a specific stem-loop structure to form, which helps to stabilize the mRNA and leads to increased translation.
- The mini cistron (in the Trc vectors) also acts to enhance translation by coding for a short gene sequence that encodes a small peptide. Since this brings the translation machinery to the proximity of the start of the gene of interest, it helps to initiate the system downstream.
- We recommend starting with a high copy number plasmid. This way, minipreps can be used directly with the Expressway system.
- Spacing between the RBS and ATG is very important for efficient translation.
- The RBS will increase the yield of protein and increase translation fidelity.
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E. coli, rabbit reticulocyte lysate (RRL), and HeLa cell lysate can all be used for in vitro translation. Our Expressway system utilizes E. coli. In general, RRL efficiently translates proteins greater than 30 kDa. The 1-Step Human In Vitro Protein Expression Kits are ideal for expressing human proteins.
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slyD is an endogenous gene product from E. coli. slyD is very Cys-rich, which makes it interact with the Lumio detection agent.
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The Expressway Lumio system incorporates the benefits of the Expressway cell-free system and Lumio technology. Using the Lumio kit, your gene of interest is fused to a Lumio tag, enabling sensitive and specific in-gel detection of the Lumio -tagged fusion protein in polyacrylamide gels without the need for staining or western blotting. You can also monitor real-time synthesis of the Lumio -tagged protein using a standard fluorometer.
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The Expressway Mini Cell-Free Expression system is designed to perform twenty 50 µL reactions or one 1 mL reaction. The Expressway Maxi Cell-Free Expression System is good for 200 x 50 reactions, which can be accommodated by 2 x 96-well plates. Both systems include the IVPS E. coli Extract, IVPS reaction buffer, feed buffer, 10 amino acid mix, methionine, DNase/RNase-free water, RNase A, T7 Enzyme Mix, 2 mL reaction tubes, and a positive expression control vector. Cat. No. K990096 also includes the pEXP5-NT/TOPO and pEXP5-CT/TOPO expression vectors.
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- 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
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A cell-free expression system is best used when working with a toxic target, as no cells are needed for protein expression. In vitro protein expression utilizes the necessary cellular components to drive expression in a single tube.
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- Begin by generating a DNA template, either by PCR or in a plasmid vector
- Purify the template
- Perform the synthesis reaction
- Analyze the sample via Coomassie staining, western blot, etc.
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We have not specifically tested for this, although we do know that the limiting factor will be the tRNA's. Because of this, simply adding more of a particular amino acid will not make a difference.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We offer the Thermo Scientific Thermal Mixer with a choice of blocks for microtubes and microplates. Please visit the product page (https://www.thermofisher.com/order/catalog/product/13687718?ICID=search-product) for more information.
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No, the E. coli strain is not a supF or supE strain, and it has very little suppressor activity. Therefore, it should be useful introducing modified amino acids.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Yes. We recommend starting out with a high copy number plasmid. This way a researcher can go right from a Miniprep kit directly into the Expressway reaction. Many of the pET vectors are low copy, and need to be concentrated before being used in an in vitro expression system.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
We have successfully scaled up the reaction to 200 ul, and find that protein expression remains linear.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Purification may be performed at 4 degrees C or room-temperature depending upon the sensitivity of the synthesized product.
1. Upon completion of incubation, remove the desired portion of reaction for His-tag purification to a clean microcentrifuge tube. Add 4 volumes of Binding buffer and vortex briefly (Add 200 µL for 50 µL of reaction). Centrifuge 5 minutes at 12,000 rpm.
2. Transfer the supernatant to a 2.0 mL tube containing 50 µL pre-equilibrated resin.
3. Incubate with shaking or mixing for 30-60 minutes.
4. Spin down resin for 2 minutes at 800 x g. Do not spin any higher or the resin will collapse and recovery will be low. Carefully remove supernatant.
5. Add 200 µL wash buffer and mix for 5 minutes.
6. Spin down resin for 2 minutes at 800 x g. Carefully remove supernatant.
7. Repeat steps 5 and 6.
8. Add 100 µL Elution Buffer and mix for 5 minutes.
9. Spin down resin for 2 minutes at 800 x g. Carefully remove and save supernatant.
10. Repeat steps 8 and 9.
Binding Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
6 M guanidine HCl (optional)**
Wash Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
15-25 mM imidazole*
Elution Buffer:
50 mM NaP04, pH 7.0
500 mM NaCl
150-250 mM imidazole*
**Depending on downstream applications, the purification may be performed under semi-denaturing conditions, or native conditions. Under semi-denaturing conditions, dilute the reaction in denaturing Binding Buffer containing 6 M guanidine HCl; then wash and elute with native buffers.
The concentration of imidazole is dependent upon the type of resin used. For Ni-NTA or ProBond resins, use 25 mM imidazole in the wash buffer and 250 mM imidazole in the elution buffer.
Find additional tips, troubleshooting help, and resources within our Protein Purification and Isolation Support Center.