Cell-Free Expression Support—Troubleshooting

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. 

You can try to reduce the incubation temperature to 25–30°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.

Protein yield may decrease as the size of the protein increases. You can try to reduce the incubation temperature to 25–30°C during protein synthesis.

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.

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.

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.

We would recommend using T7 RNA polymerase (Cat. No. 18033019, 50 U/µL). Use 1–1.5 µL in a 50 µL reaction system.

Unfortunately, this may result in a loss of activity.

Please see the following recommendations:

• Check your DNA template for configuration (ATG initiation, tags, stop codon, in frame, etc.).
• Ensure that the DNA template is pure.
• Do not purify your DNA using gel purification, as this can inhibit the protein synthesis reaction.
• Ensure that the correct amount of DNA template was used; we recommend 10–15 µg of template DNA in a 2 mL protein synthesis reaction.
• Use a thermomixer or incubator with shaking for the protein reaction.
• Ensure that the correct amount of MembraneMax™ reagent is used.
• Try different “feed” schedules—e.g., add one-half volume of feed buffer to the sample (i.e., 25 µL feed buffer to 50 µL sample) 30 min and 1 hour after initiating protein synthesis.
• If expressing a large protein, try reducing the incubation temperature to 25–30°C during synthesis.
• The protein may be degraded; limit incubation to <2 hr and minimize handling in between reaction steps.

Yes, you can incubate your reaction up to 4 hours to try to increase protein yield, although this may result in the formation of polydispersed micelles. Since 2 hours of incubation typically results in more than 75% expression for most proteins, longer incubation is usually unnecessary. You can try to increase the incubation temperature to boost the rate of translation or decrease the temperature to allow the protein more time to fold properly. Please note, do not decrease the temperature below 24°C, as this is the transition temperature of the DMPC lipid bilayer of the MembraneMax™ reagent. Modifying the feeding schedule may help with your synthesis reaction. For example, you can initiate the reaction with 50% reaction volume, then add 25% reaction volume of feed buffer twice at 30 min intervals. Lastly, you may scale up the reaction to obtain larger quantities of membrane protein, as protein yield is reaction/volume-dependent.

All-trans retinal could have been bleached; ensure that the all-trans retinal is stored in the dark, as it is photolabile.

Improper protein folding could cause low activity. Try reducing the incubation temperature to as low as 25°C during protein synthesis. Additionally, post-translational modifications of your protein may be required for activity. The Expressway™ E. coli slyD-Extract will not introduce these modifications (such as glycosylation or phosphorylation). Lastly, synthetic proteins may require co-factors for complete activity. Ensure that the required co-factors are added to the protein synthesis reaction.

The scaffold protein from the MembraneMax™ Reagent will migrate as a 28 kDa band on an SDS-PAGE gel.