Cell-Free expression system is widely used in protein production. Some proteins that are difficult to express and purify in living cells, such as membrane proteins, GPCR and ion channel proteins, are often produced in cell-free expression systems. In addition to e.coli and wheat germ cell-free expression system, Profacgen developed a high yield cell-free protein expression system using tobacco cell lysates. Tobacco Cell-Free Protein Expression is mainly used to produce antibodies, antigens, enzymes and membrane proteins. As it is an animal-free expression system, the protein expressed and purified from tobacco cell-free system is safer and more suitable for animal in vivo testing.
Cell-free protein expression systems(CFS) (Conjugated transption and translation systems):
- Subcloning: cloning of a target gene (either PCR product or a plasmid);
- Transcription Reaction-1: constructing an expression vector containing T7 or SP6 strong promoter, tobacco mosaic virus 5’ leading sequence with GAA (Translation enhancer), a florescence protein and multiple cloning site (MCS). Then insert the target gene into the expression vector and perform in vitro transcription after interacting with the transcription mixture (DNA template, SP&RNA Polymerase, NTP Mix, Transcription Buffer and RNase inhibitor).
- Transcription Reaction-2: the obtained mRNA was used as template for translation in Translation Buffer (containing 20 kinds of amino acids, ATP and GTP). There are other three translation methods that can be used in non-conjunction with the instrument, including Bilayer Reaction Method, Repeated Batch Method and Filter-Feed Method. Among them, the most widely used is the Bilayer Reaction Method.
- Product Analysis: we can analyze the fluorescent signal, the quantity the fluorescent signal and luciferase activity.
Tobacco Cell-Free Protein Expression is a famous due to its own advantage.
- Comparing with other CFS, higher expression level;
- Shorter cycle that can get the target protein in 24 hours;
- It has the function of post-translational modification of plants.
- Higher purity recovery of protein (with tags or no tags) which is used in some proteins with low expression level and membrane proteins with complex structure.
- The protein can be directly used in animal experiments with animal origin free.
Our scientists can optimize the codon preference of specific proteins according to your needs, construct vectors and express instantaneously in tobacco cell-free protein expression systems, detect the transcription of specific proteins at the RNA level, and detect the expression of target proteins. We always put your needs first, and if you have any questions about the results, we can help you verify them. Profacgen is your ideal partner, if you are interested in our services, please feel free to contact us for more information.
 Buntru M, Vogel S, Stoff K, Spiegel H and Schillberg S. (2015). A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates. Biotechnol Bioeng. 112(5):867-78.