Dear all,
I am expressing a protein having a MW of 32kDa by using pET21d+ vector and Rosetta gami strain.
I have tested several temperatures between 16 and 20°C and IPTG concentration between 0.3 and 1mM for the expression and all these experiments gave me always the same result. A protein is expressed in high quantity but it has not the expected molecular weight because SDS-PAGE and MS analysis give a MW of 40kDa, 8kDa more than the expected 32kDa. NO BAND appears at the desired MW.
I have observed also other interesting phenomena:
The first thing that one is led to think is a problem with the sequence, but one of the ORF in 5'->3' direction on my plasmide is in agreement with the expected protein and 6xHisTag. A problem with a unrecognized stop codon is also possible and could be able to extend my protein sequence, but the stop codon is TGA, that is the suggested one for E. Coli strains. In addition, it is after HisTag as it has to be. However, also by considering a wrong recognizing of the stop codon is hard to think that 8kDa are added to the original protein.
By considering these phenomena, I am thinking that the protein that I would express is toxic for the used strain (it is not a pLysS or a pLysE) and that the band at 40kDa is an other protein expressed by the strain in this particular condition.
Is this plausible or can other explanation be possible?
What do you suggest to perform the expression in these cases?
Thanks for your suggestions.
Danilo
I suggest you identify the protein by MS (peptide analysis) if the 40 KDa protein not is your protein you are wasting your time.
Hi Benny, if your protein is hydrophobic or basic it binds more quantity of SDS, consequently her mobility in PAGE will be lower than expected by the amino acids composition. MW in PAGE of an expressed protein is not a problem if you do not know the original protein.
I suggest to streak your positive clone on a plate with the opportune antibiotic before to start the growth in liquid. Try to do a western blot using a his-antibody (the best one is H1029 Sigma-Aldrich Monoclonal Anti-polyHistidine antibody) with lysates of 1) IPTG-induced and 2) not-induced cultures, along with lysate from E.coli with 3) empty plasmid and 4) without plasmid. If the lanes with lysate from your positive clones are black (positive, does not matter the MW) your protein is there, if still your western blot is without signals (negative), there is some problem with your construct.
Rosetta cells are useful when you are expressing big proteins (>70kDa) or with unusual codons. Have a check of the codon usage, but keep in mind that is very difficult obtain good quantities of COOH-His tagged proteins. I prefer to use pQE30 .
If your protein has long hydrophobic stretches could be toxic in E.coli. You can realize it looking at your culture. In fact cells lyse (DNA appears in the culture) and the mass of your culture does not increase.
Remind that during the screening phase you can use a denaturing protocol of purification, containing 8M urea or 6M guanidine-HCl, your Ni-NTA chromatography will work and the buffers will inactivate the proteases. Let me know and good luck
Hi Danilo!
Did you checked if your protein is being expressed or not? i mean, did you compared the induced cultures with the not induced?
If your protein is being expressed, and that checks are positive, the 8 Kda increase could be possibly addressed to metal binding of your protein. Did you performed an ICP-MS? Did you checked the isotopic pattern of you Ms spectrum?
I would also agree that you first need to be certain you can really express your protein.
We have had a similar problem, protein migrated at different place, did not bind to the nickel column even with a his tag in its ORF. We thought it is our protein but after quite some time of fruitless experiments we concluded we cannot express the protein at efficient levels.
So, first try to confirm the band identity with mass spectrometry.
Thanks for your suggestions.
However, as in my description, the MW of the protein is confirmed by MS spec (ESI-MS of the whole protein). Thus, it is not a problem of migration but of actual MW of the expressed protein.
Regarding the wester blotting with His-antibody, it has been already performed (only on the purified protein) and blot does not appear. A wester by Histag antibody on denaturated protein could suggest if the lack of blot is due to an unavailable Histag or if His tag is not there.
Finally, I have tried to compare IPTG-induce and no induced and the band appear only after induction. I have not an ICP-MS available thus, I can not assess the problem of the metal binding. Do you think that in the presence of SDS and by heating the sample at 95deg for 5' the metals can actually keep bound the proteins?
Danilo
In that case, definitely try to confirm the protein identity by in-gel digestion and MS-MS (or at least peptide mass fingerprinting).
Another explanation, such as frameshift on the mRNA is much less likely the cause.
this is a good news! your protein is expressed. There would be a plethora of possible explanation. of course metals could be still bound to your protein, even after the denaturation step. In fact if the ammino acid sequence of your proteins contains particular cysteine/methionine regions, it's likely that there your protein would bind metals, since these amino acids tend to form quite stable metal complexes, even after the denaturation steps. you can see more information here
http://pubs.acs.org/doi/abs/10.1021/bi0478392
the fact that the anti-his antibody does not respond to the protein, could be a clue for a possible aggregation issue of your protein. It means that the epitope specific for the antibody recognition may be hidden and not easily reached. did you try to treat the protein with some chelating agents before the MS analysis or the SDS PAGe assays? probably they could help to strip the metals possibly bound to your protein. Or before SDS-Page you can try other more powerful denaturing agents, such as Urea.
If the MW is correct, then something is amiss with your sequence. Re-clone into a pGEX vector or similar and use a decent fusion tag (His tags are a pain). You can always cleave off the GST if needed.
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