What is "Tsukuba System"?

The "Tsukuba system" refers to a transient protein expression system in plant cells, which can accumulate 4 mg/g fresh mass in 3 days using Nicotiana benthamiana. As shown in the figure on the right, we were able to express a large amount of proteins in a short period of time, compared to the magnICON system, which has been considered to be a best transient protein expression system in the world.

4 mg/g fresh mass may not sound like much, but when converted to mL = g, our system can produce a comparable amount or protein, compared to heterologous protein expression system such as E.coli and Brevibacillus. 

How did it become possible to express so much?  By combination of the geminiviral rolling circle replication system (see here) with a double terminator, we established high protein expression system. The only single terminator probably causes interference of transcription due to the read-through (see here), but the double terminator is thought to have eliminated the interference of transcription, resulting in high expression of protein. 

If you want to produce a large amount of protein, it is best to express it in Nicotiana benthamiana, which produces about the same amount of protein of interest as Rubisco. It is capable of production 40-50% of total soluble protein.

However, the Tsukuba system is also useful if you want to check localization in the plants you are using. The system can be applied to other plants, such as Solanaceae (eggplant, tomato, and pepper), Cucurbitaceae (melon), lettuce, and orchid. Although the amount of protein is not as high as that in N. benthamiana. But protein of interest can be expressed in several kinds of plants.  For your information, protein of interest was not expressed in tomatoes and lettuce.

I also tried this system to wild tomatoes, and legumes (Lotus japonica, soybean, and common bean). In the case of legumes, it is easy to see the expression in the pods. In the case of roses, I was not able to express protein of interest in neither petals nor leaves. I do not know why it was not applicable in roses.

We are also working hard to build case studies by using our system. If it becomes possible to overwhelmingly produce a variety of proteins, I believe that more people will be willing to try using our system. So far, we have succeeded in producing Bet v 1, an pollen allergen of birch tree, and in expressing PMab-2, an antibody against RAP tag. These results have been introduced as a press release.

[Bet v 1作出に関するプレスリリース] [PMab-2に関するプレスリリース]