I expressed three proteins(ex.A, B and C) in enterotoxigenic e.coli except C with 0.5mM IPTG. (C is expressed in other bactera) and then I checked it using commassie brilient blue staining and western blotting. (Ab. his-probe antibody)
A protein size is 63kDa and finally I found the target protein is expressed well.
B protein size is about 50kDa, but there are 2bands, one is about 63-65kDa and the other is 50kDa.
last protein C size is 58kDa, but there are many bands, one is about 63-65kDa and others are about 40~60kDa.
all bacteria express 63-65kda size protein.(there are no-band or leaky band before IPTG treatment) so I wonder protein A is the protein what I want or not.
they have only one something in common and that is they use same carrier protein. but still I don't understand why they express same size protein.even the size 63-65kDa is not the target size of protein B and C.
Does anybody knows about this happen? how bacteria express protein which is not my target? how can I understand this problem?
Dear Garam,
Have you tried running a negative control (which will NOT be containing any of your proteins). An ETEC strain transformed with an empty vector, when induced with IPTG should give you an answer.
Abhishek
thanks for your answer, Abhishek Sen. Well, I didn't try running vector-only as a negative control in ETEC. But I tried it in BL21 and there was no band in 63-65kDa. As you say, I will try again with vector-only control. but if there is no band, what should i do? Do you have any idea to understand this situation?
Can you show a gel picture and give some details of the methods you used to obtain the samples?
I understood that the three types of proteins expressed have same sizes in 63-65 kDa in common. You should purify the target protein first using his-tags. First, some modification or denaturation condtion for SDS-PAGE can affect the protein migration speed. Also, disulfide-bond shuffling during the fractionation on SDS-PAGE gel can affect protein migration speed. Also, other contaminating proteins can affect the protein migration speed. In my knowledge, the purifcation will be helpful to identify the exact size of the target protein. Authentic sizes of the protein can be revealed during purification step. Check the antibody specificity in Western blotting. There are similar cases in HPV E7 protein expressed in E. coli. The theoretical size of E7 protein is about 11 kDa. However, they have more higher sizes (19 - 20 kDa) when they expressed in E. coli.
Dear Garam,
Since I am working on ETEC as well, I can anticipate that if the 63 - 65 kDa protein is not coming from the ETEC host strain it is most definitely coming from your vector, after being induced. Do share your findings, should be interesting to know.
Abhishek
Hi,
when I induce expression of recombinant proteins with IPTG I do get non-specific induction of other proteins in my expression cell line. I think this is a relatively normal phenomena, and it is okay because the non-specific proteins are a different size to my target protein. If however you can't differentiate them, I would suggest blotting with an monoclonal anti-(your target) antibody to find your protein or using another expression cell line where this non-specificity doesn't occur
J
Use western blot to check if the 65 kDa protein contain His Tag. If it does has His-tag, then there maybe an inefficient translation stop codon and you have a leaky translation that goes too far. If these 65 kDa protein do not have His-tag, then you have nothing to worry about, they will go away after Ni-NTA column purification.
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