I have produced a recombinant enzyme in two different e. coli strains and, since this is a metalloprotein, I am adding the metal in the moment of induction. I get higher yields of protein production with one strain, compared to the other and also, higher activity levels (even higher than the native protein). Is this somehow explainable?
Metal ion is a cofactor for this enzyme.so time of protein folding is very importants in presence of the metal or absence of the metal
The fact that some strains of E.coli behave in different ways than others is self-evident, I think. For what reason do we have strains that are especially fit for expression?
Information on how exactly the strains differ may help you in understanding your result.
Depens on how much you really care to explain it, rather than being happy that it works better with another strain you have...
Enzymes are environment specific. You change pH or Temperature or ionic concentration, the activity changes. Similarly, there is clear difference in the cellular cytoplasm for different strains. You may look at the metal update in both strains. This may explain.
It is explainable, but you do need to provide a bit more detail. For instance, what do you mean by 'yield'? Two different strains may differ in their final density during cultivation, hence yields are different simply because of differences in starting wet weight. Expression levels may also differ, that's another possibility. Also, what do you mean by 'higher activity levels'? Are you referring to specific activity?
Depending on your strains you may have different levels of successfully assembled protein. If, for example, one strain only folds 10% successfully, or only loads the cofactor into 10%, whilst the other strain is successful for 100% of the soluble protein, you may still get the same amount of protein but only 10% is active. This is aside from the absolute amount of protein you obtain, which is incredibly strain dependent (codon optimisation, folding enhanced, etc.) or condition dependent (temperature, amount of induction, etc.).
With these different strains I get different amounts of protein (498 mg and 2,26 g) The activity I refer is specific velocity.
I can think of a few prominent influencers here: 1) intracellular processing/expression, 2) sample matrix composition, and/or 3) handling & storage conditions of specimens. You may have an intersection of one, two, all three, and beyond this.
Different strains of bacteria can have different doubling times, nutrient consumption, and rates for transcription, translation, and modification of gene products. Internally, these factors affect the product mass and initial activity. [Ellis O'Neill pointed this out above.]
If the enzyme is then harvested from the media, the pH, temperature, ionic strength (as Dr. Kangueane mentions above) would definitely influence enzyme conformation (and hence activity)....particularly as the culture ages and the media gets more acidic, O2 declines, etc. If you are lysing cultures and purifying from there, I don't know what would happen.
Then storage. Depending on the enzyme and its usual optimal activity conditions versus those encountered after collection, enzyme activity could rapidly decline (or even increase!) with storage...it's well known in clinical lab science that serum/plasma enzyme activity measurements generally aren't reliable after long-term cold storage. You may be able to measure mass still, however.
One final consideration - are you sure you accounted for excess of any required cofactors when you measured activity? With the >4-fold difference you report in enzyme mass, one size doesn't fit all for reagents unless there is significant excess.
You ststed that you get "With these different strains I get different amounts of protein (498 mg and 2,26 g) The activity I refer is specific velocity." this is a big difference. You did not give data on the relative specific activity so I will try to guess.
One problem with over "over-expression" is missfolding, this could yeld lower specific activity. This is many times inhanced by to fast expression (if this the case I will assume the 2.26g strain will give less specific velocity). Try lowering the concetration of the inducer and/or inducing at lower temparture (down to 18C).
A second issue (for metalo-protein) might be metal concetration inside the cells. Could it be that one strain allow more metal in? This is testable by ICP of cell pellet.
Just some thoughts, if you supply more detailes we might suggest better ideas...
Yoram
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