We have cloned a wide range of Gram-positive enzyme genes (cytoplasmic or secreted in the original host) in various E. coli cells and always can detect the activity around the colonies on plate or in the culture supernatant. There is also no difference if the signal peptide is present or not - E. coli is not supposed to actively secrete foreign genes anyway. The majority of the recombinant protein is in the fraction with the cells (after centrifugation), even for those proteins having a Gram-positive leader peptide - so what is the heck? Just isolate the protein from the cells.

What type of plasmid did you use for transformation of gene of interest (recognised sulfatase) with DH5-alpha competent cell?Normally, there will be a known signal peptide for desired expressed gene if the enzyme activity of that functional protein gets a significant result. Enzyme leakage may occur when the membrane of cell get rupture and no longer functional from being destructed by the foreign substances such as the enzyme leakage in human organ like liver, heart muscle and so on.

Cytoplasmic sulfatase belongs to Lysosomal enzymes,Whether not appear in the right [Pseudomonas aeruginosa] cells, part of the cells [E. coli DH5]within the substance may be dissolved,so enzyme leakage from E. coli cultures.

The above is my personal conjecture!

may be toxic for your cells which results in lysis? did you perform a growth curve with and without the plasmid or /and with without IPTG (where is your T7 polymerase coming from, is it a DE3 strain?)

The bugs could be locating the sulfatase to the periplasmic space. In that case you would see more enzyme in the sup. with time and less activity in the pellet.

I suspect the answer lies between the following parameters: (I) age of the culture when the activity is observed in the culture supernatant, (ii) the host cell which in this case has many mutations that might render it inherently leaky, and (iii) cytotoxicity which is inducing cell lysis. Another question to ask is what % of total activity is in the supernatant?

Protein leakage generally occurs when cells are exposed to substrate limitation. Please see the following publication :

Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect.

Frank Delvigne, Alison Brognaux, Frédéric Francis, Jean-Claude Twizere, Nathalie Gorret, Soren J Sorensen, Philippe Thonart

Biotechnology journal. 05/2011; 6(8):968-78.

The problem also occurs in LB medium, so no substrate limitation, its a fools paradise for a bacterium...

We have cloned a wide range of Gram-positive enzyme genes (cytoplasmic or secreted in the original host) in various E. coli cells and always can detect the activity around the colonies on plate or in the culture supernatant. There is also no difference if the signal peptide is present or not - E. coli is not supposed to actively secrete foreign genes anyway. The majority of the recombinant protein is in the fraction with the cells (after centrifugation), even for those proteins having a Gram-positive leader peptide - so what is the heck? Just isolate the protein from the cells.

Maybe your protein sulfatase is toxic for DH5-alpha cell.

PLEASE DO bellows procedure for testing toxicity:

Check your protein existence in your cell and media with western blotting.

If your protein exists in your cell check this protein is in inclusion body or dissolved.

You can find that your protein is toxic or not?

Cytoplasmic sulfatase belongs to Lysosomal enzymes. Protein leakage generally occurs when cells are exposed to substrate limitation.

i think may be of come from lysed Dh5-alpha cell present.

First I would suggest changing strains, DH5alpha is RecA- and hence in an overnight culture you may have nearly 50% dead cells that will be lysing. Use a RecA+ strain to minimize that. Otherwise it will also correlate to culture density - age. You will always get some leakage but with a good strain and exponential cells it should be less than 10% for most proteins.

Many thanks for all the comments and suggestions. I should have mentioned that the proportion of sulfatase activity being "leaked" into the media is low (about 1%). It was interesting to read that Wolfgang Schwarz has observed similar extracellular activity with heterologous expression.

The suggestions that the potential toxicity of the over-expressed sulfatase could be at work are valid: I need to use low IPTG concentration (10 micromolar) to achieve good growth and expression of the sulfatase. (NB. I am not using Studier's T7 system; rather, a pJexpress plasmid from DNA2.0). 10-fold more IPTG slows colony growth by about 3 or 4-fold.

The reason I am curious about this "leakiness" is that I would like to develop a selection strategy that relies on extra-cellular activity to select new sulfatase variants. I'd like to improve the enzyme toward new substrates, but could end up selecting enzyme variants that have unintended consequences (e. g. greater leaking by increasing toxicity and cell lysis, without improving enzyme activity per se). I think I'll proceed with a method that uses the inherent extracellular activity, but include a secondary screen that examines activity of crude cell lysate.

Thanks again to you all for your interest and taking the time to write.

Use Rsetta E.coli to prevent leakage expression

I've also apparently observed this small leakage of cytoplasmic proteins from E. coli colonies - because they were showing unexpected extracellular activity (e.g. when expressing B. subtilis APase in E.coli it was slowly reacting with BCIP in the agar and making the colonies blue despite being produced as cytoplasmic: (Fig.S3) https://istina.msu.ru/download/3838841/1ghpEm:9swTzk3s9_PH94lIGbI8g5B8lQw/).

I've thought it's some kind of oddity but judging from this tread this leakage of a very small % of a heterologous protein is actually a common feature.

Interestingly, in case of two different proteins (40-60 KDa), activity remained in the colonies (substrate assays have colored the colonies only, not their surroundings) while another one (~19 KDa) was also forming large halos around the colonies (=diffusing away).

Though, I'm not fully sure it's a simple leakage from dead cells: it can be more complicated and involve live cells.

Bradley James Stevenson How did your screen work out?