Hi Oliver,

Most of the time, a "spacer" between a His-tag and a target gene was put into the vector in order to fit in a multiple cloning site (MCS) or a protease cleavage site. IMHO, a "flexible spacer" isn't necessary (except for the functions stated above). In many crystal structures, the termini of the proteins are flexible with high B-factors (or simply can't be resolved). If you think an N-term His-tag is disrupting the structure or function of your protein, put it at the C-term instead.

Best regards,

Arthur

if you are going to clone your gene into a vector already having a pelB-leader, you don't need to add any sequence, and don't modify the sequence immediately after the pelb sequence either. The pelb sequence is the signal sequence for your target protein transported into the periplasm where the pelb is  processed off  and your target protein folded. Any modification could lead the process failed and your target protein will not proper folded.

If you want to customize your own vector you can just copy the pelb coding sequence form a commercialized vector.

Adding His tag in front of the pelb could lead to malfunction of pelb, in most cases adding after pelb is ok if not disturb the processing site.

Since in most cases you don't know if there is any flexible sequence either at the N-ter or C-ter end of your target protein, it is no harm to add some spacers to keep the tag more flexible for binding to the resins when you add his tag (or any other small tags) in either ends.

So I generally use pET 22 b vector (with pelB-sequence) in the user manual of the vector I didn't find any information if I need a "spacer sequence" between pelB and my protein or not and between pelB and N-Terminus of my protein are anyway amino acids given by mcs-site. One of my pET22 b protein constructs has very low expression efficiency in contrast to other "good" working pET22 constructs. Also I only use codon and secondary optimized genes. Furthermore I use  E.coli codon usage plus cells. I differ expression temperature, IPTG concentration and shaking conditions, but with no improvement. So my suspicion is, that the additional amino acids between leader and gene forms in my case unfavorable structures, which lowers the export efficiency in periplasm. Also it is possible, that proteases could hydrolase the protein in periplasm, when the folded protein has the wrong structure and as a consequence of this I see a very low expression of my aim protein.

Thank you for your ideas!

pET-22b carries a C-terminal His tag. You can alter the length of the "spacer" between pelB and your target by choosing a different restriction site. Have you got any model of the protein structure? Are either terminus predicted to be flexible or not?

Sometimes, protein may be insoluble or toxic to the cells. What is the origin of your protein? Just because it's codon optimised for E. coli doesn't mean it will fold properly in E. coli. Expression strains such as BL21 are protease deficient, so it's unlikely that your protein got broken down by proteases in the bacteria.

1) Expression:

The protein without pelB-Leader can be well expressed in E.coli. (in pET21 vector)

2) Structure:

at N-terminus starts a alpha Helix at AA-Position 3-20.

at C-terminus is also a alpha Helix and a 3/10-Helix until 3 AA to end of the chain.

http://www.uniprot.org/uniprot/H8WR05

I didn't find an information for the flexibility in the structure paper of my protein. It is possible to predict (by a webtool) the flexibility of the parts of a protein ?

In the crystal structure, both termini are pointing away from the rest of the protein. In theory, putting a tag at either end shouldn't interfere with the structure. The B-factors of the crystal structure seem to indicate that they should be flexible relative to the rest of the structure as well. But then again, putting the pelB right before the alpha helix at N-terminal may introduce other undesirable effects (I am just saying "may" here, not saying it's highly likely). If it expressed well in the cytoplasm, why are you trying to export it to the periplasm? Actually, this page from EMBL ( https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/improving_protein_solubility/ ) suggests that expression yields are much lower when you use pelB leader sequence. If you really want to export it into the periplasm, there are also other vectors with different signal peptides.

Sorry that I can't be of more help.

Yes I know that fact, that expression is lower with pelB-leader.

But for other proteins that express well with pelB-leader i can very easily purificate proteins, which are secreted in periplasm. After an osmotic shock, the concentration of other e.coli proteins is very low and after a single NTA-chromatography step often a single protein band is seen by SDS-PAGE.

Yes another signal peptide might be able to improve export, but when I don`t has a reason/theory why pelB should doesn`t work, I will not try further secretion-expressions.

But thank you for your very detailed answers!