Nature Protocols Discussion Forum forum: topic
This is a public forum
Fusion protein
siti fuzi
Saturday, 19 July 2008 04:35 UTC
I’m doing mutagenesis..so in the early stage,I try to fuse between signal peptide from E.coli and extracellular protein from fungus(I’ve already delete the signal peptide site of this fungus).Then i’m using vector pET22 and host BL21 for the expression.But the problem is,when I tried to express the recombinant protein without the E.coli signal peptide as a control, the protein still can be detected outside the cells. Is it possible or is it because of eukaryote protein that I used?I can’t find any reference regarding this problem. Do anyone has faced this problem before?I tried to use other commercial signal peptide, but it doesn’t work either(protein can still b secreted outside the cell)..Thank u
-
Replies
-
I’d double check your sequencing to make sure there’s not a signal sequence in the plasmid itself. Looking at the documentation, there’s “an N-terminal pelB signal sequence for potential periplasmic localization” in the pET22 vector.
Otherwise, you could try running his sequence through these:
http://www.cbs.dtu.dk/services/TatP/ (signal sequence independent secretion signal)
or
http://www.cbs.dtu.dk/services/SecretomeP/ -
Siti,
Below are a couple of thoughts for you to consider.
The solution to your problem might depend on what your objective is. Proteins can be purified from the culture medium, if you ultimate goal is to obtain purified protein, so having the protein accumulate in the medium may not be so bad. Let me know and I can offer some advice in this regard.
Some proteins, when hyper-expressed in E. coli, can lead to lysis. A decrease in the OD post-induction would be indicative of lysis. Reducing the level of expression might help if this is the case.
If you are certain there are no cryptic signal sequences in your constructs, then my suggestion is to try alternative host strains first before revamping your expression vector. In our experience BL21 isn’t always the best strain. I suggest HMS174 or BLR.
Is there any protein accumulating in the cytoplasm? If so, what is the relative fraction and might this be enough to accomplish your objectives? What is the viability of the cells post-induction?
Good luck.
-