Hi Peter,

If your protein of interest (POI) is insoluble, adding pelB-leader sequence is not going to help. You must use a solubility tag like GST, MBP or SUMO proteins. In the worst case scenario, you can purify your POI in denaturing conditions, and try to refold it later.

Let me know if you have any more questions.

Best,

T.

Hey Peter,

try other temperatures e.g. 15 up to 30°C after induction of protein expression. The incubation can took place over night. Lower temperatures should lower expression rate and could avoid aggreations. When you use an pelB Leader, try a shock with TES-medium, high sugar concentrations can also support protein solubility.

http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/index.html

Check the size of your protein: It is possible to get periplasmic inclusion bodies, the protein in these would have the leader sequence cleaved off, see http://aem.asm.org/content/52/5/1157.full.pdf+html for some nice EM pictures

You could combine both the secretion into periplasmic space and charms of SET (solubility enhancing tag) by using plasmids for the fusion with periplasmic versions of DsbA or DsbC. It significantly reduces the yield but might rescue solubility. If youyr protein has disulphide bridges thioredoxin would be a fusion protein to try first for cytoplasmic expression. Worked for me:

http://www.ncbi.nlm.nih.gov/pubmed/18983922

The simplest and fastest options are to reduce Temperature 30/20/15°C or/and to reduce the IPTG concentration ;-) . If this doesn't, you can try to change tags/organisms or vectors.

I do like Peter but I use phoA as leader sequence. It also form inclusion bodies in cytoplasm. 

My protein is 56-kda, it too big or not? Should I try other leader sequence?

You could try using E. coli Origami cells to help with disulphide bond formation. For more information: https://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

Actually, it is also possible to get periplasmic inclusion bodies, as observed, for example, with poorly folding antibody scFv fragments - in contrast to cytoplasmic inclusion bodies, the leader sequence has been cleaved off upon secretion.

• @ Annemarie Honegger May you, please tell me how to remove signal peptide in the cytoplasm. or which is the best signal sequence to use?

The signal peptidase associated with the sec expression system (LepB,LSPA) is responsible for removing the signal sequence during the translocation process.

https://www.qiagen.com/us/shop/genes-and-pathways/pathway-details/?pwid=286

If the signal sequence gets removed, the protein has undergone translocation. It is exported and aggregates only after export to the periplasm. On the other hand, if your protein still contains the leader sequence, it most probably has not been exported, but aggregates in the cytoplasm, either because your expression levels exceed the capacity  of the sec postranslational secretion system or the secB chaperone system is not capable of keeping the protein from folding/aggregating before it can be exported.

See: S:Schlegel et al., Optimizing heterologous protein production in the periplasm of E. coliby regulating gene expression levels (Microb Cell Fact. 2013; 12: 24.) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605120/

You can try to target your protein to the co-translational SRP pathway ( DsbA-derived signal sequence) https://www.bioc.uzh.ch/plueckthun/pdf/APpub0297.pdf

If you want to keep the protein in the cytoplasm, you express it without a signal sequence. Generally, the chances of getting soluble proteins are higher for slower expression (lower plasmid copy number, weaker promotors, lower temperature, weaker induction). Coexpression of chaperones and disulfide isomerases may also help. However, the effects of these parameters are weak compared to those of improving stability and folding efficiency of the expressed protein itself.