Technology

Overview

We have three area of technologies.

Template:

Conventionally, 3’UTR side was required to be around 1200 bp in order to synthesize mRNA and its stability. But it prevents from attaching C-terminus tag by PCR. Thus, vector construction and cloning used to be a must.

We found approximately 50 bp works great as 1200 bp. By adding this short sequence to the 3′ UTR, C-terminus tag is also possible by PCR, without any cloning or vector construction.

We are providing tool, excel VBA macro file, to designing primers very easily. You can copy and paste ORF sequences and select which terminus you want to add the tag. Then, tool calculate Tm values with nearest neighbor or GC% method for several forward primers and reverse primers. You can select the best possible pair of forward primer and reverse primer, and copy and paste the forward primer and complement sequence of reverse primer.

download : 1stprimeraidv2.zip

The PCR enzyme has an extension time of 30 sec per kb and is 80 times more accurate than Taq polymerase. In addition, you do not need to investigate cycling conditions when primers are  longer than 20 mer (Tm value > 63 ° C).
Furthermore, compared to the conventional 3-step PCR cycle, there is less non-specific amplification / smear that causes troubles in gene amplification, transcription, and translation. And PCR time goes to  1/3 of the time compared to conventional 3 step cycle.
pcr prducts 
Also, in addition to peptide tag such as His, HA and FLAG, large molecular tags, GST and/or Halo can be with PCR. (the protocol will be up soon)
Translation:
Play Video

In addition to transcription factor, secreted protein and and others, optimization of the translation solution derived from wheat germ has made it possible to synthesize membrane proteins soluble. Also, liposomes is not necessary for expression of membrane proteins. Lipids derived from wheat germ (membrane fragments) are adsorbed to the transmembrane domain of membrane proteins and soluble. Lipid from wheat germ will work as chaperon or membrane to make it soluble.

Simple and Affordable:
Simple Mixture

There was concern about precipitation of amino acids prepared for translation reaction, but 20 kinds of amino acids are converted into one solution separately with high concentration by our technology. Therefore, preparation of the translation reaction solution is simply mixing the provided wheat germ extract solution with the amino acid mix. For translation reaction, you can use a simple mixing method in which mRNA obtained by transcription product is added and incubated. Please refer to the  video on the support page, which includes the preparation process.

These three  technologies make it possible to easily synthesize the following proteins, which were conventionally difficult to synthesize:

Expression Example

Proteins from 10 kDa to 200 kDa

Various proteins from around 10 kDa to around 200 kDa, from kinase to photo receptor,  can be synthesized.

Phosphorylation enzymes

Phosphoenzymes involved in immune responses, which have been difficult to synthesize, have also been synthesized with activity.

Cytotoxic protein

It is also possible to synthesize cytotoxic ribosome inactivation proteins (rRNA N-glycosidase, tritin), which were difficult for recombinant expression in the past.

Secreted protein

Secreed protein and/or cytokines such as granulocyte colony stimulating factor(GCSF, CSF3) can be synthesized.

Transmembrane receptor kinase

Transmembrane receptor can be synthesized and is soluble.

MATE (multidrug and toxic compound extrusion)

MATE, which is ubiquitous in living world and important transporter , can be synthesized as soluble protein.