Cell-Free Expression Support—Getting Started

• 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.

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.

• 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

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.

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. 

pEXP3-DEST (supplied with K9900-70 and V960-03) allows you to fuse Lumio™ and 6xHis tags to the N-terminus of your protein of interest using Gateway technology. pEXP4-DEST (supplied with K9900-90 and V960-04) allows you to fuse Lumio™ and 6xHis tags to the C-terminus of your protein of interest using Gateway technology.

The Lumio™ recognition sequence is a small 6 amino acid sequence of Cys-Cys-Pro-Gly-Cys-Cys. The Lumio™ detection reagent binds this recognition sequence with high specificity and affinity, causing a bright fluorescent signal for real-time protein production analysis and immediate in-gel protein detection.

The Lumio™ Green Detection Reagent has a maximum excitation at 500 nm and maximum emission at 535 nm. This allows for detection of the Lumio™ fusion proteins using a UV transilluminator equipped with a standard camera or a visible light laser-based scanner.

While real-time detection of Lumio™ tagged proteins can be performed, the signal strength does not correlate to protein expression levels, so performing in-gel detection is recommended in addition to real-time detection.

Precipitate your proteins with acetone to remove background smearing, then add the Lumio™ Gel Sample Buffer (4X) and Lumio™ In-Gel Detection Enhancer for polyacrylamide gel electrophoresis. 

slyD is an endogenous gene product from E. coli. slyD is very Cys-rich, which makes it interact with the Lumio™ detection agent.

• 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.

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.

Both vectors contain a T7 promoter, RBS, and T7 terminator with spacing and sequence configuration optimized to allow for high levels of protein expression in the Expressway™ system. The pEXP5-NT/TOPO vector contains an N-terminal peptide containing the 6xHis tag and a TEV recognition site to allow production of a recombinant fusion protein that may be easily detected and purified. The pEXP5-CT/TOPO vector contains a C-terminal tag containing the 6x His tag to allow for production of a recombinant fusion protein that may be easily detected and purified. Protein yields can vary significantly depending on whether the recombinant protein of interest is expressed as an N- or C-terminal fusion, and therefore, both constructs should be tested.

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.

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.

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.

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.

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.

These Gateway-compatible vectors both have a T7 promoter and a T7 terminator. The pEXP1-DEST vector contains a ribosome-binding site (RBS). If a RBS is present in your gene or as part of the entry vector, pEXP2-DEST can be used (since it does not have a RBS present). In addition, the pEXP1-DEST vector has an N-terminal 6xHis-Xpress fusion tag, while the pEXP2-DEST vector has a C-terminal V5-6xHis fusion tag for convenient detection and purification of your recombinant proteins.

No, the proper machinery for glycosylation is not present in these extracts.

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.

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.

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%.

We recommend incubating the protein synthesis reaction at a temperature range from 30–37°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°C); however, protein solubility generally improves with incubation at lower temperatures (i.e., 30°C).

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.

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 for recommended amounts of labeled and unlabeled methionine.
• Heavy metal–labeled methionine: Use selenomethionine (Budisa et al., 1995; Doublie, 1997; Hendrickson et al., 1990) to produce labeled protein for use in X-ray crystallographic studies. See Performing the Protein Synthesis Reaction on page 21 of the manual 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.

E. coli cells do indeed contain some chaperone proteins used for protein folding. However, extra chaperone proteins were not added to the extract.

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.

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.

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.

No, unfortunately, the machinery for glycosylation is absent in these extracts.

If needed, we recommend the addition of PMSF (final concentration 0.5­1.0 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 Pafablock C (final concentration 0.1–0.2 mM AEBSF) in your transcription/translation reaction. Both are serine protease inhibitors.

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 on page 22.

Yes, we offer our MembraneMax™ Protein Expression Kits, which combine novel membrane protein solubilization technology with the Expressway™ system for in vitro expression of soluble membrane proteins from template DNA in a single scalable reaction.  

The HN stands for His-tagged NLP. We recommend using the MembraneMax™ HN kit if you would like to express your membrane protein in its native state (untagged) or if you wish to use a tag other than 6xHis. We recommend use of the MembraneMax™ Protein Expression Kit if you would like your membrane protein to be expressed as a fusion protein containing an N- or C-terminal polyhistidine tag.

Please see the table below:

NLPs are discoidal particles of approximately 10 nm in diameter consisting of a proprietary formulation of a scaffold protein ring that encloses a planar DMPC lipid bilayer.

The following DNA templates can be used:

• Supercoiled plasmid DNA (recommended to obtain the highest yields)
  
• Linear DNA
  
• PCR product

Please note, for proper expression, all templates must contain the T7 promoter, an initiation codon, and a prokaryotic Shine-Dalgarno ribosome-binding site (RBS) upstream of the gene of interest.
Additionally, the pEXP1-DEST, pEXP3-DEST, pEXP4-DEST, pEXP5-NT/TOPO, and pEXP5-CT/TOPO vectors, as well as any other vector system that would allow the expression of polyhistidine-tagged proteins, must be used with the MembraneMax™ Protein Expression Kit, and not with the MembraneMax™ HN Protein Expression Kit. See page 6 of the manual for details. Any expression vectors containing a sequence for the C-terminal polyhistidine tag, including pEXP4-DEST and pEXP5-CT/TOPO, can be used with the MembraneMax™ HN Protein Expression Kit, only if:

• The polyhistidine tag is not used for the purification of the membrane protein, or
• The gene for the membrane protein of interest and the sequence for the polyhistidine tag are separated by a stop codon in the expression construct.

You may use a variety of methods to purify your DNA template including commercial DNA purification kits or CsCl gradient centrifugation. Do not gel purify your template, as purified DNA solution obtained from agarose gels significantly inhibits the protein synthesis reaction. See page 12 of the manual for more information. 

The following may be added to the reaction mix to increase solubility:

• Triton X-100 (0.5–1%)
• Dodecyl-maltoside (0.025–0.05%), CHAPS (0.1–2%)
• Octylglucoside (1–2 mM), Brij 35 (0.5–0.1%)
• Octyl-gluco-pyranoside (1–1.5%)

The lowest incubation temperature is 25°C, because 24°C is the transition temperature of the DMPC lipid bilayer in the MembraneMax™ Reagent.

Typically, R&D reports supercoiled plasmids giving higher yields in our labs. However, linear constructs work well if:

• They contain the right promoter, spacer, ribosome-binding site, ATG, and terminator sequences
• Purified PCR products are used and 2–3 µg purified DNA is used for expression
• The linear construct is not isolated using gel extraction

The most important suggestion would be to ensure that your reaction is mixing thoroughly throughout the incubation period.  We recommend using a thermomixer incubator set to 1200 rpm or a shaking incubator set to 300 rpm. Do not use a stationary incubator, as protein yields can be reduced by up to 30–50%. Additionally, incubation of the protein synthesis reaction should be carried out at a temperature range from 30–37°C, with higher protein yields generally obtained with higher temperatures. Lastly, the reaction can be incubated for longer than 2 hours (up to 4 hours) to increase protein yield, though this may result in the formation of polydispersed micelles and protein degradation.  

The height is 5 (± 0.5) nm determined by atomic force microscopy, while the diameter is 10 nm. 

The pEXP5-NT/CALML3 vector is used for expression of the N-terminally tagged human CALML3 protein. The pEXP5-CT/bR vector is used to express bacteriorhodopsin (bR). Together with its cofactor, all-trans retinal, bR can be used as a positive control for membrane protein expression through a colorimetric control assay. This control assay is typically performed in parallel to the membrane protein synthesis reaction to indicate whether the components of the MembraneMax™ Protein Expression Kit are functional. Successful expression of the correctly folded, functional bR in the presence of its cofactor causes the reaction mixture to change from a pale yellow color to a pink color in approximately 15 min. If this reaction is not observed, you can try expressing CALML3 in order to help troubleshoot your experiment.

A negative colorimetric assay followed by positive expression of CALML3 fusion protein would implicate a degraded MembraneMax™ Reagent, while the lack of CALML3 protein expression would point to the degradation of the reagents in the Expressway™ Expression Module.

For a 100 μL protein synthesis reaction, use 1 μg of template DNA (plasmid or linear DNA). For a 2 mL reaction, use 10–15 μg of template DNA. For optimal results, purify the DNA template before use.

Successful expression of the correctly folded, functional bacteriorhodopsin (bR) in the presence of its cofactor, all-trans retinal, causes the reaction mixture to change form a pale yellow to a pink color in as little as 15 min. Longer incubation (up to 2 hours or more) results in higher bR yields and stronger color change.