Hi
I am currently studying the expression of the TEV protease recombinant protein, and unfortunately, I have encountered a somewhat illogical problem during my work. I would greatly appreciate it if you could help me based on your experience and knowledge.
One of my colleague’s previously used the soluble fusion tag “GST” for expressing the TEV protein. In their design, they were able to express TEV/GST protein in SHuffle strain by using the pBAD A series vector under araBAD promoter and ori: p15A.
However, the protein was totally expressed into inclusion body. In order to optimize that project, we decided to use the previous backbone, however with an alternative tag based on an article by Dr. Yutaka Kuroda entitled " A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity " Consequently, GST fusion tag was replaced with SEP tag, incorporated in C-terminal. This article claimed that this tag significantly enhances the solubility of the TEV protein.
It should be noted that Dr. Kuroda used the pET15b vector under T7 promoter in their design.
After changing the solubility fusion tag, the integrity of the target fragment was confirmed by Sanger sequencing. In spite of the confirmation of critical elements within the expression vector, no protein was expressed in Shuffle (induced by Arabinose at 30 and 16 C for 4 and 18 h, respectively), even into inclusion body forms. I have included the gel images of my colleague's vector and my own below for your reference. (The expected size of TEV/SEP is ~ 29 kDa, while GST/TEV is approximately ~58 kDa.)
Furthermore, since we don't need to purify the TEV protein in my project, this protein is not fused to His- tag.
the important question for us at this moment is the lack of protein expression by the vector.
Can you please help me why we have not any band in our SDS-page gel of our recombinant protein?
DEar Hamed Moslehi
2 questions:
1) Do you need the presence of the SEP tag at the N-term?
2) Are you sure that there was not a frame shift in the link between SEP and TEV?
in my past experience TEV can be produced quite well with both His and MBP tag in Rosetta strains using a lower induction temperature (eg.25°C or 30°C) in the soluble form.
In this contect ou can buy from Addgene (cost about 100$) one of the vectors developed by DAvid Waugh with the TEV S219V stabilized mutant
https://www.addgene.org/browse/article/301/
good luck
Manuele
Hi Dear Manuele
In fact, my protein and SEP tag is connected to the C-terminal TEV based on the similar work that Dr. Kruda and his colleagues did in 2018.
I am completely sure that there has been no frameshift, and the start to stop coding sequence has been read by Sanger (three times with no mutations, including the promoter, RBS, and terminator).
Thank you for the information you provided about your experience, but for some reasons, I can not use any vector, and if I decide to purchase a new vector, I will have to change several parts of it, which will be a difficult task.
I am almost certain that there is no mutation in my vector and protein structure, and as I mentioned, the sequencing of the gene confirms this, but the question remains: why is the protein not expressed.
- Badges
- Science topic
- Similar topics
- Papers