Yes, it is expected that protein yield resulting from linear templates is diminished compared to its circular plasmid version. A decrease of 10-30% is considered to be within the normal range.
myTXTL requires high quality template DNA, which should be free of nucleases (DNases, RNases) and inhibitors of the TXTL machinery (e.g. EDTA, ethidium bromide, SDS, Cl- ions, ethanol). Preparation of plasmid DNA with standard commercial kits usually involves sample treatment with RNase, which may not be completely removed during downstream processing. Thus, we strongly recommend subjecting the prepared DNA to either a commercial PCR clean-up kit or standard phenol-chloroform extraction and ethanol precipitation. Ideally, template DNA is in deionized, nuclease-free water. Please note, introducing Mg2+and K+ ions can compromise the kit performance, as they are extremely critical for transcription and translation, and are optimized in the master mix. Plasmid DNA prepared with a ZymoPure system purification kit or obtained from a commercial vendor can generally skip this extra purification step. Linear templates generated via PCR require just a single PCR Purification kit clean up (or magnetic bead cleanup) step or no clean-up if the PCR reaction is being diluted directly into the myTXTL reaction (be sure glycerol in the reaction is 0.1% or lower). See kit manual for additional recommendations.
Apart from standard biochemical methods like Coomassie-stained SDS-PAGE and western blot analysis, the great advantage of cell-free protein production is the open-system environment which allows the direct quantification and/or analysis of protein functionality in an activity assay without purification. Alternatively, for some activity assays downstream processing via affinity purification may be needed (if an affinity tag is present). If you choose SDS-PAGE analysis, you can either take a small sample (1-3 µL) directly from your TXTL reaction, or – to reduce background signal – precipitate proteins with TCA/acetone or ammonium-acetate/methanol following a standard protocol.
It is very important to avoid condensation of water on the lid of the reaction tube as it considerably increases the concentration of myTXTL reaction components and can lead to poor or irreproducible performance. Incubation in an incubator and water bath/Lab Armor beads is best. Water facilitates a faster heat transfer than air and a water bath shows low temperature fluctuation, which should – combined with a closed environment with constant temperature surrounding the entire tube – lead to higher reproducibility and yield. myTXTL reactions also work well on an Eppendorf ThermoMixer with a lid.
We rcommend using the heavy and light chain templates at an equimolar concentration of 5 nM each whether they are plasmid or linear DNA templates.
Yes! Parameters that influence protein production efficiency are:
- DNA design (promoter strength, position of affinity tag, TXTL elements)
- DNA purity
- DNA concentration
- Incubation temperature, time, and vessel
- Presence of folding helpers, chaperones, oxidizing agents
and should therefore be evaluated for optimization. Please also see our recommendations on these topics in the current myTXTL manual for your kit.
Consider if your recombinant protein requires co-factors like heavy metal ions or coenzymes to be functionally active. Those should be present during protein synthesis. Additionally, a low concentration of mild detergent (e.g. Triton-X-100, sodium dodecyl maltoside, or CHAPS) can be added to the reaction as well as molecular chaperones. Please note that the myTXTL system cannot introduce post-translational modifications like glycosylation or phosphorylation to your protein. Reducing the incubation temperature might help to prevent aggregation of the nascent polypeptide chain and to promote proper protein folding.
The positive control plasmid in the myTXTL Antibody/DS kit expresses the Gaussia Luciferase Dura protein which has 5 disulfide bonds. It is visible on an SDS-PAGE gel without purification when compared to a negative control reaction. Its activity can also be assayed using the NanoLight GLuc GLOW assay (NanoLight Technology; cat no. 320-50) and quantified using the GLuc standard sold separately (NanoLight Technology; cat no. 321-100).
The myTXTL Pro Kit is best for rebooting bacteriophage and some modifications to the reaction mix may be needed for rebooting your phage, please refer to the manual for detailed suggestions on phage rebooting.
An additional application for the myTXTL Pro kit is that it can be used to reboot bacteriophage from a genome input. Several phages have been shown to replicate in the myTXTL system and they generally benefit from the following modifications:
1) The addition of 0.3 mM each of additional dNTPs (for replication of DNA genomes) and 0.5-4% PEG 8000 can be helpful for optimal production of phage. These components along with your genome, 9 uL of Pro Master Mix, and water if needed should add up to 12 uL and be assembled in a 1.5/2 mL tube. Larger reaction volumes may require shaking as described in the manual for larger scale reactions. Incubate overnight as in our protocol.
2) You may also need to optimize the phage genome concentration if it is not already published. 0.25 nM has worked well for T7 but T4 was optimal at 1 nM. In part this is dependent on the quality of the genome. It is extremely important that the phage genome is of very high purity, free of contaminants and of high integrity.
3) Commercially available T7 phage genomic DNA can be used directly in the Pro Master Mix as a positive control (we have used DNA from BocaScientific: https://bocascientific.com/310000-86-detail).
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