I have expressed a gene encoding 15 kDa protein in E. coli BL21RIPL cells. I dissolved the pellet in 1M tris, pH7.5, then sonicated , centrifuged. now the supernatant(soluble fraction) and the pellete (Insoluble) are giving me different sizes for the expressed protein. The supernatant has smaller protein of approx 13.5 kDa while the pellet shows 15kDa band(= the size of protein in crude fraction). Kindly cite reasons why is this happening.
Hi, did you had a negative control? you should have also induced empty vector and compared with your induced clone bands. Sometime it might be leaky expression from the vector used. Which expression vector did you used? what is you expected protein size?
the only vector clone does not have a band induced at this size. i have used pET28a(+). I do not get any protein expressed in so much conc in uninduced and no vector clones.
Do you have gel picture? Did you induced your empty vector as same as you clone?
But you might be knowing approx size of protein? else as you have his tag you can try western blotting and confirming the expression of your protein.
If you only know sequence of your protein, you can predict size of protein using online available ExPasy compute PI/MW tool
i know the size given by expassy 15 kDa. i hv used the empty vector and expressed bt there is no protein expressing as such amount at this size, though E. coli own proteins are there. i hv attached a gel pic with my ques
I am sorry I did not noticed gel pic earlier. Still I have following ques :
1) Lane 1 is it from empty vector or your clone?
2) Lane 3 why did you dissolved supernatant in buffer? we usually direct add loading buffer and run on the gel. Pellet is re suspended in lysis buffer before loading.
To me it looks your induced protein is in pellet and that's normal with E.coli expression system.
Kindly find the attachment I have drawn arrows which I feel are same bands.
To further confirm best would be to do a western blotting.I have a feeling expressed protein is in pellet.
I hope you loaded equal conc. of protein in all wells.
It is possible (although maybe unlikely) that after sonication periplasmic proteases can access the soluble protein and trim it (especially if there is a degron or other recognition sequence near the end of the protein). I would explore that possibility by looking at the sequence, adding (more) protease inhibitor, or testing expression in strains that lack major periplasmic proteases.
I have seen a similar problem before in one of the labs I worked in and the use of knockout strains help determine that this was the cause of the problem.
Have you checked for activity of this protein. If you have access to mass spec you can get the mass. Some time we see variation in how the soluble protein in supernatant and the protein isolated from the pellet run differently especially on non-reduced SDS.
I agree that it is possible that following sonication your protein is partly degraded by endogenous e. coli proteases. However, BL21 lacks several major proteases (Lon and OmpT) so this would be quite unlikely in this strain. Could you be more specific about your protein? Is it a membrane protein, or a soluble protein? Did you check whether your protein contains a signal sequence? If that is the case, than it is conceivable that signal sequence is clipped off during transport and this may explain the band of a lower MW in the soluble fraction. This fraction contains the cytoplasm and periplasm, while the insoluble fraction comprises debris and inner and outer membrane.
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