Expressway™ Tag-On-Demand™ Cell-Free E. coli Expression System *DISCONTINUED* - FAQs

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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- 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.