I suspect your protein is not getting to the periplasm and is most likely forming inclusion bodies (evidenced by it being in the cell pellet). Is there a reason you need it in the periplasm (disulphides perhaps)? You may need a change of plan and attempt recovery from inclusion bodies for instance.

I don't think sonnication is going to change anything. You may also have to consider that P450s are generally membrane proteins (if I remember correctly) - perhaps they are getting stuck in the membrane rather than going through into the periplasmic space. Detergents may help?

Dear Kadir Aslan

sonication is quite strong and it is not so simple to perform selective periplasmic extraction, lysis of total cells is hijgly probable.

If periplasmic shock provide you low protein expression while total cell lysis show that an high fraction is in the pellet, i suppose that the most of your protein is expressed as insoluble form, maybe in the IB and probably the issue is more related to protein unfolding than cell lysis approach and therefore you have to optimize you expression condition more than the lysis one.

some questions:

Why did you perfrom periplasmic extraction? Is it your protein rich of S-S bond? If yes! Did you add DTT or an other reducing agent in your lysis buffer when you perfomed sonication? THe presence of a reducing agent in the lysis buffer is able to improve protein extraction? Did you performed expression test even at lower temperature? Lower temperature are able to improvre the amount of soluble protein?

best regards

Manuele

Manuele Martinelli Gary James Hunter

Dear Prof. Martinelli and Prof. Hunter,

Thank you very much for your insightful comments. My target proteins are type of and close to plant‐derived cytochrome P450s cloned with a pelB leader (pET-22b backbone) so that they are exported to the periplasm. My rationale was to minimise aggregation and obtain a fraction that is relatively free of cytosolic contaminants while preserving activity.

In my current osmotic-shock protocol doesnt have any nlysozyme and detergents. From the literature and my own trials, yields without lysozyme are only acceptable for very soluble proteins; for most targets, recovery is low. This matches your point about solubility being the main bottleneck. Although I cannot yet rule out inclusion bodies, the evidence is mixed: with auto-induction at 18 °C the culture shows almost no product at 24 h but a clear band at 48 h, and an eGFP control cloned in the same vector fluoresces strongly. After sonication (65 W, 20 % amplitude, 0.2sec pulse in 1 sec, 3 and 5 min) I see little release at 3 min and good release at 5 min, yet the pellet remains fluorescent. Because GFP fluorescence normally disappears in true inclusion bodies, I suspect incomplete lysis rather than heavy aggregation. I hesitated to test 50–80 % amplitude out of concern for enzyme activity; eGFP is therefore my proxy while I optimise the parameters before assaying the scarce P450 substrate.

Is it still a sensible strategy in this context, or would you recommend returning to a cytosolic expression system and tackling solubility with lower temperature/co-expression of chaperones instead? I do not yet have structural data; is there a straightforward screen you recommend to check for essential disulfides before deciding between cytosolic vs. periplasmic production?

Ultimately I only need a reasonably active enzyme (not necessarily 100 % yield) that I can produce in bulk, store, and assay under different activation conditions. Do you have any practical suggestions?

Thank you again for your time and guidance.