Several good answers have been given already. In addition to potential problems with protein folding, improper secretion to the plasma membrane, or inhibitory post-translational modifications, the protein of interest may also be associating with inhibitory protein complexes. You could try using a high salt concentration (300-500mM) in your lysis buffer to dissociate any inhibitory protein complexes.

Is there any reason why you don't add protease inhibitors to the lysis? This may cause problems with protein degradation.

Are your lysis conditions in the cells to harsh? This can cause problems, when protein complexes dissociate.

What are the differences between your experimental conditions between E.coli and your mamalian cells?

Western blot only confirms that your protein is expressed, but not if its active (since SDS-Page works with completely denaturated proteins).

Whether the protein is heterologous?(meaning that the mammalian cells don't have it). If so, it is something to do with folding or the active site being internalized.

If your protein is undergoing post-translation modification in mammalian system but it is not in E. coli expression system.

As you had mentioned that your are dealing with membrane protein, I feel strongly to do something with sub-cellular localization.

As Christian mentioned western blot will confirm the presence or absence of protein.

I hope this will help you.

chandru

Is the expression level of your protein similar in E.coli and mammalian cells? If it is much lower, your assay may be not sensitive enough.

If you use fluorescent substrates, they may bind to other things in lysate which could compromise detection. You may supplement the mammalian lysate with lysate of expressing E.coli . This will show whether you have smth. in the reaction mixture that inhibits detection. Mislocalization and misfolding are also likely. Immunofluorescence could give you some clues.

Most probably, the protein folds differently in bacterial and mammalian cells. This is especially true, if your protein contains several Cys-residues. Incorrect closure of disulphide bonds might influence aggregation state of the protein. You need a more detailed analysis of the correctness of the fold of your protein.

Try a native gel and do an insitu staining with the substrate. Load both the E coli and mammalian protein. You could also try separating the membrane and the cell lysate and check. Could give you some clues.

Several good answers have been given already. In addition to potential problems with protein folding, improper secretion to the plasma membrane, or inhibitory post-translational modifications, the protein of interest may also be associating with inhibitory protein complexes. You could try using a high salt concentration (300-500mM) in your lysis buffer to dissociate any inhibitory protein complexes.

Many good suggestions already.

Microsomal sample preparation might be an easier way to optimize your assay since your target protein is membrane protein. Microsomal preparation help enrich your protein (if comparably too low than E. coli), or simplify sample environment with removing cytosolic contents including protein degradation machineries.

But this way might not work if problems are due to lacks of protein folding, necessary post-translations etc.

Best. .

The expression level in your mammalian system is likely to be very low compared to E coli and your assay may not be sensitive enough. The western blot is a very sensitive technique compared to fluorometric assays based on peptide cleavage. Still, you may try the following to maximise your chances. First, you have to be sure you inhibit all unwanted proteolysis. For this you have to lyse the cells in a buffer containing a complete protease inbhibitor cocktail (you may try several to find out which one does not inhibit your protease of interest). Second, because you are working with a membrane protease, you may try to enricjh your activity by making a membrane prep (ie 20,000 g or 100,000 g centrifugation from a post-nuclear supernatant) and solubilise this with various detergents (in buffer containing inhibitor cocktail) to select the most effective. Third, when you do the enzymatic assay, you have to set up reactions in parallel with and without an inhibitor specific for your enzyme. There will always be unwanted degradation of the substrate, thus to determine the enzyme activity you have to subtract values in presence of inhibitor from those obtained with no inhibitor.

Good luck!

Thank you all for the responses so far! I am new in this field, so I also don't fully understand all the answers, but I will figure it out or I will just ask :).

Where I work now, they normally don't have problems when not adding protease inhibitors, but I will definitely use that from now on!

I've tested total protein amount in both HEK and e coli and made sure I added similar amounts to the assay, but unfortunately that didn't help. Of course the concentration of protein of interest might be much lower anyway. There is however a limited volume available for the cell lysate in the assay.

Regarding the inhibitor suggestion: there are no known specific inhibitors, that's exactly what we're trying to find. We did do a screening and got some positive hits on the e coli cell lysates. These results we want to validate in the mammalian cells, but there (obviously) we ran into these problems. So it still is a good suggestion, although I'll not be 100% sure it's a specific inhibitor.

So my questions at this moment are:

- How can I make sure the lysis is efficient and still have the end product as concentrated as possible? So far I've been using 10 cm plates and resuspended these cells in just 200 ul buffer to try to get it as concentrated as possible. I also tried 2 ml to make sure the lysis worked, but that didn't help as well (but of course also less protein then).

- My protein apparently is mainly ER membrane bound. Does that work the same in regard to your suggestions as a 'normal' membrane bound protein?

- What is the best (read: easiest) way to analyze the protein folding?

- It has been suggested to add high salt to the lysis buffer to dissociate any inhibitory protein complexes. My protein is only active when bind to its co-factor; will high salt dissociate this complex as well? If so, I should not use it.