Troubleshooting Guide for NEBExpress™ Cell-free E. coli Protein Synthesis System (NEB #E5360)

Problem Possible Causes Solution
Control protein is not synthesized   Kit component(s) were inactivated Store NEBExpress S30 Synthesis Extract and the Protein Synthesis Buffer (2X) at –80°C. Minimize number of freeze-thaw cycles (if necessary, aliquot components).
Nuclease contamination Wear gloves and use nuclease-free pipette tips and microcentrifuge tubes.
T7 RNA Polymerase was absent in the reaction Make sure to add T7 RNA Polymerase.  
Control protein is synthesized, but the target protein is not present or present in low yield. RNase contamination Commercial mini-prep kits are a useful tool for the preparation of template DNA but are often the source of introducing RNase A to the in vitro protein synthesis reaction, make sure RNase Inhibitor has been added to the reaction.
Template DNA design is compromised Ensure the sequence of the template DNA is correct and in-frame. Make sure the DNA template contains the T7 terminator or a UTR stem loop to stabilize the mRNA in order to increase yield.
Non-optimal regulatory sequences and/or spacing may adversely affect translational efficiency. Translation initiation is critical, secondary structure or rare codons at the beginning of the mRNA may compromise the initiation process.
If tolerable to the target, the addition of an adequate initiation region (e.g., first ten codons of maltose binding protein) may help. Alternatively, using PCR to modify the 5′ end of the target gene may eliminate secondary structural elements or rare codons.
Certain eukaryotic proteins might require modifying the gene sequence for expression in a bacterial system (codon optimization, addition of tags to enhance solubility, removal of transmembrane domains, etc.)
Template DNA is contaminated Inhibitors of transcription or translation may be present in the DNA. A simple mixing experiment (control DNA + target DNA, compared to control DNA alone) will reveal whether inhibitors are present.
Do not use DNA purified from agarose gels as they often contain inhibitors of translation (e.g., ethidium bromide). Residual SDS from plasmid preparation protocols is another common contaminant. Re-purify the DNA using Monarch® Plasmid Miniprep Kit (NEB# T1010) or Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030).
Make sure to add RNase Inhibitor to the reaction. The supplied RNase Inhibitor will reduce the effects of RNase contamination that may be present after using commercial plasmid prep kits.
Template DNA concentration is not optimal The concentration of template DNA is important to maintain the balance between transcription and translation. Too little template reduces the amount of actively translated mRNA while too much template results in the overproduction of mRNA and overwhelming of the translational apparatus. 250 ng of template DNA is recommended for a 50 μl reaction. Optimization with different amounts of template DNA (e.g., 25–1000 ng) may improve yield of a particular target protein.
If UV absorbance was used to calculate the concentration of the template DNA, be aware that RNA or chromosomal DNA will also absorb UV light. If the sample has significant amounts of RNA or chromosomal DNA, the actual amount of template DNA may be lower than the calculated amount. The 260 nm/280 nm ratio should be 1.8. Running a sample of template DNA on an agarose gel can determine if it is the correct size, has been degraded, and/or if it contains other nucleic acids.
Target protein synthesized but the full-length product is not the major species Translation initiation and/or termination is not correct   The system can express large molecular weight proteins; however, obtaining full-length product requires proper initiation and termination. Internal ribosome entry sites and/or premature termination can produce truncated proteins.
Target protein synthesized but it is inactive or insoluble Incorrect folding Incubating at lower temperatures (down to 16°C), for longer periods of time (up to 24 hours), can help solubilize proteins that otherwise are synthesized in an insoluble form.
Alone or in combination with changes in incubation temperature, supplement the reaction with PURExpress Disulfide Bond Enhancer (NEB #E6820). Add 2 µl of Enhancer 1 and 2 µl of Enhancer 2 per 50 µl of reaction.