Dear Margot,

I do not know what type of heterologous protein you are planning to produce, but at least for some recombinant antibodies the cytoplasmic targeting is sufficient to cause leakage to the surrounding media, allowing the bypass the RIPA-step altogether. You of course end up losing  lot of the product, but it depends what is necessary, cost effective etc.

I have done batch cultures for scFvs and Fabs with PelB that allows periplasmic expression. There, the leakage differed from clone to clone and the size and charge seemed to play a role.

This review might help you out choosing suitable system for soluble secretion from E.coli.

https://microbialcellfactories.biomedcentral.com/articles/10.1186/1475-2859-4-1

Hope this helps.

-Juha

Dear Juha,

Thank you very much for your answer.

Right now I am “playing” with the HlyA secretion tag fused in the Cter of the protein, and I cannot detect my protein in the media.

So my first idea was to check if it was produced as inclusion bodies or in the soluble form. Could you share with me the buffers and protocol you are using to answer this kind of question?

I am looking into the review you sent.

Thanks a lot.

Margot

Dear Margot,

As far as I know, inclusion bodies can be solubilized only in very harsh conditions (high concentration of GdmCl or urea), so I would not worry abut them too much. You could always use ultracentrifugation at 100,000xg to get rid of the aggregates. If your lysed pellet is sticky, you could add some DNase in order to cut the bacterial DNA.

I hope this will be useful.

Rafal

Secretion requires specific secretion fusion tags, absent these you are looking for excretion into the media by leaky bacterial cells. This can be achieved via media sometimes or through the use of specific strains engineered to leak. See this paper and refs therein to explore these strategies.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241728/

Also the T7 promoter system (commonly used in BL21(DE3)) is hugely strong and commonly produces more soluble protein when subtly induced (as in Studier autoinduction media, at low temp. etc) or just with basal (non induced) expression. 

Inclusion bodies can also be a short cut to soluble protein if in vitro refolding can be explored.