There are a number of variables that are important to test:

1. Signal sequence we routinely test the native signal sequence (where appropriate), pelB, OmpA, DsbA, TolB and MalE. All are E. coli derived except PelB which is from E. carotovora. We thus target the sec and SRP pathways. We see target protein dependent preference for different signal sequences.

2. Strain: we have had a couple of proteins that have failed/largely failed in B834(DE3) but have worked nicely in Rosetta2(DE3).

3. Media: we routinely always compare an IPTG induction in a rich media against autoinduction media carried out at 20C and 25C respectively. To fine tune expression it can be helpful to add glucose to the former and vary the induction temperature for both. It should also be noted that rich media isn't always the best for high levels of expression and we have also seen better expression of some proteins in more minimal media.

Hi Stefan, many thanks for your reply!

As far as I know, the TorA 'pathway' is checking for correct folding (maybe you want to compare http://juwel.fz-juelich.de:8080/dspace/bitstream/2128/109/1/leben_14.pdf). I am not sure if an introduced human protein might fold correctly in E. coli and thus be processed correctly by the TorA pathway. Anyway, using a TorA signal is tempting in the age of synthetic biology, as one may easily to construct the sequence in a manner that it may easily be exchanged with another signal cassette if required.

What do you think?

Im using pET26b vector (PelB leader) in BL21 Codon Plus strain and it works! but the yield is low! so if anybody knows how to up my yield in the membrane that would be a great great help!! Thanks and good luck!

Whatever system you are using: Secretory pathways are complex and more prone to be compromised by small changes/mutations than "ordinary" expression systems. So it easily happens that after a few days on a plate the "great producer" turns into a dwarf. My standard rules for most expression systems plus some extras for secretion to obtain max yields:

- transform freshly

- keep the promotor well repressed

- cultivate on selective and rich plates at low temperatures

- grow liquid cultures at 25-30°C (whatever your strain allows)

- secretion is energy dependent, add glucose (max 1%), maltose or other carbohydrate (depends on the expression system)

- aerate the cultures well, use flasks with baffles or even Fernbach-flasks, but never use too high shaking rates (-> shearing, stress, extra energy waste)

- the best tool for secretion is a well buffered and aerated Bioreaktor, in my thesis, out of desperation, I built a simple device on my own, because the institute's reactor was not always available. There are some single use devices around that probably can be reused a few times.

a leader signal as the one used by pMal plasmid from NEB for secretIon of proteIns fused to maltose bIndIng proteIn Into perIplasm..

Alternatively,

scFv expression can be directed to either the oxidizing environment of

the periplasmic space, under regulation of the pelB leader sequence

(Padiolleau-Lefèvre et al., 2006), or to the culture supernatant using the

α-hemolysin (HylA) systemof E. coli (Fernandez et al., 2000; Fraile et al.,

2004).

In the past I expressed the soluble fragment of a yeast enzyme in the periplasm of E.coli using the ompA signal, however I got a double band which suggested incomplete removal of the tag. In any case, I managed to improve the yield by changing expression conditions, in particular the medium. In my case, folding was aided by longer expression at lower temperature, and the yield was better when I used M9-CA medium enriched with glycerol and the rec. enzyme cofactor (compared to using LB or TB). Efficient isolation of the periplasmic fraction also required some fine-tuning of the purificaiton protocol. Best of luck!

There are a number of variables that are important to test:

1. Signal sequence we routinely test the native signal sequence (where appropriate), pelB, OmpA, DsbA, TolB and MalE. All are E. coli derived except PelB which is from E. carotovora. We thus target the sec and SRP pathways. We see target protein dependent preference for different signal sequences.

2. Strain: we have had a couple of proteins that have failed/largely failed in B834(DE3) but have worked nicely in Rosetta2(DE3).

3. Media: we routinely always compare an IPTG induction in a rich media against autoinduction media carried out at 20C and 25C respectively. To fine tune expression it can be helpful to add glucose to the former and vary the induction temperature for both. It should also be noted that rich media isn't always the best for high levels of expression and we have also seen better expression of some proteins in more minimal media.

It is difficult to predict which signal peptide will be the best: dont' forget that in an organism, the signal sequence has co-evoluted with the begining of the mature protein. In your project, you use an prokaryotic signal sequence with an eukaryotic gene. The cleavage efficiency will depend of the first residue of the mature protein: some residues with positive charges, in particular lysines into the 10 first positions, may be a real problem for the cleavage by the signal peptidase. Consider also that a too large production (driven by a strong promoter) may produce inclusion bodies even in the periplasmic space. some E. coli strains are able to form the disulfide bridges in the cytoplasm (See the Novagen pET manual). Good luck

Colette

I would second Louise's suggestion to consider several signals, and to optimise the expression conditions carefully. Whilst pelB has worked well for me on several projects, it is likely that a eukaryotic protein will need careful tuning.

I would be wary of using the Tat pathway: this is really designed for proteins that require complex cofactors (e.g. iron-sulfur clusters) that have to be assembled in the cytosol, and hence requires that the protein is folded correctly in the cytosol. You are looking to express in the periplasm because your protein has disulfide bonds or some other PTM that makes cytosolic expression unattractive (right?), so Tat is unlikely to work for you - if it did, then you would just express in the cytoplasm!

Colette's suggestion to consider the cell lines (Origami etc.) that allow disulfide bonding in the cytoplasm is also worth following up if that's your issue. I've got reasonable yields of a disulfide bonded human protein out of Origamis in the past.

Be aware - about 50-60% of human proteins never express solubly in E. coli, no matter how hard you try. Extracellular proteins are usually worse than intracellular ones, as they are almost all glycosylated and otherwise modified. Be prepared to try Pichia or refolding.

Good luck!

Nic

Can I ask you why you want to express your proteins in the periplasm? This space is smaller than cytoplasm and you need to find an appropriate signal sequence. If you are interested by the oxidizing properties of the periplasm, you may know that some engenereed E; coli strains can be really interesting. Rosetta gami are really fine for expression of disulfide bonded proteins in the cytoplasm! Moreover, these cells are well suited to express eucaryotic proteins in E. coli

Hi Pauline,

basically, I try to follow and modify an existing protocol and I'd also take advantage of the oxidizing properties of the periplasm. In addition (according to that existing protocol), I'd like to access the periplasmic proteins by hypotonic shock lysis and hope to have less unwanted stuff and less protease activity.

Do you think it is not worth the potential hassle and cytoplasmic expression is good enough (maybe even gives substantially higher yields)? I do not need active (i.e. correctly folded) protein, as long it is recognized by an antibody.

Expressing in the cytoplasm will normally give you better yield, but it is true that you have more problems with proteases presence!

Your antibody has been generated using a folded or unfolded proteins, or peptides? It is also impotant to be sure that it will recognize your proteins !

I agree with Louise about the desirability of trying both the Sec and SRP pathways. See these references:

SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli.

http://www.ncbi.nlm.nih.gov/pubmed/18504019

Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display.

http://www.ncbi.nlm.nih.gov/pubmed/16823375

Appraisal of translocation pathways for displaying ankyrin repeat protein on phage particles.

http://www.ncbi.nlm.nih.gov/pubmed/20800093