Hi all,
A strange phenomenon is troubling me now.I am very confused and ask for a help...
Please look at my attachment.
I did a 3 proteins' co-expression test assay to check my protein expression.So I transformed two plasmid (the plasmid pET28b-ProteinA and pCDFDuet-Protein B-Protein C in the attached figure) into BL21(DE3) bacteria cell and then coated on plates containing Kana ad Strp antibiotics.
After incubated over night @37C ,I picked 3 single colonies into 3ml LB media which added Kana and Strp antibiotics . 37C,220rpm, for 5h ,OD reached 0.6.1ml bacteria solution of every colony was transfered into another tube and was added 1mM IPTG to induce proteins expression for 3h @37C 220rpm.
At last I take every bacteria solution to run SDS-PAGE gel.But the result shows protein A have 2 different expression bands（showed as red and yellow arrows ）,which confused me toughly.
Why does the same plasmid expresses two different protein bands in coomasier blue stain?
In fact, plasmid pET28b-proteinA came from 1 single colony and sequencing was right.In addition,ptotein B and C expression are all consistent（showed as blue arrows ）.
So,who can explain the expression difference between colony 1# 2# and 3# .
Did another plasmid contaminate? The difference of expression activity of colonies? or SDS PAGE made it ?
This is my first time to face this strange phenomenon in expression test assay.Who can help me?Your reply maybe will release me.
Thank you for your attention.
Hi Dali CY Hu,
my suspicion would be that you have a contaminating plasmid. This might be at a low level in the original prep and you been unlucky to pick a colony with was not clonal. Did you re-streak the original cloning colony? Have a very close look at the sequencing waveform, you might be able to see a second underlying sequence appear when it changes from the vector to insert.
There's also a possibility that you have a gene that is susceptible to rearrangement. In which case there might always be a sub-population that's generated - however I would expect this to produce a mix within each expression colony.
You could extract the plasmid from the expression clones and resequence to see what you have got.
-Richard
Hi,@Richard Christison
Thanks for your answer.
Yes,contamination is one of possible explanations .But... it is unbelieveable... because I always pick mono-colony to do plasmid miniprep and it never show any error at all .
you are right,this might be at a low level in the original prep...I am so unlucky...
I have not re-streaked the original colony.But I am amplifing and culturing colony 1# and 3# to expect purification of ternary protein complex A-B-C.If the 3rd test colony is wrong,it will not purify complex well.Do you think it is feasible to exclude the wrong colony through this method?
best regards
Hi Dali CY Hu,
in most cases picking a single colony is not a problem, however I have had mixed colonies before. You can get a colony that is from two E.coli cells each transformed with plasmid that are together on the plate. They will look like a single colony after they have expaned. In many cases they could have the same construct so to all intents and purposes they are the same. However you can have cases where they are different. This can be difficult to spot sometimes if one is in the majority. This is why best practice is to restreak - I like many don't generally do this, unless I have a reason to - but it's good to understand the risks in not doing so.
As for screening by the protein complex, I think the answer would be dependent upon what the wrong protein is. If it's a truncation then It might still form the complex, you would know how likely this is better than me. If you can't determine it from the size on the SDS page, I think that mini-peeping the plasmid from the expression strain and resequencing it would be the best way to get an answer. Generally you can get OK quality DNA from BL21(DE3) but if it's problematic transforming it back into a cloning strain will help.
-Richard
Contamination is a possible explanation, but you are sure it is not the case. I think is possible that the cloned sequence encoding two different ORF, but Why only in the colony 3? Are you sure that the culture conditions were the same for the 3 colonies?
The other possibility is that the recombinant protein has been degraded; I recommend you repeat the experiment very carefully, maintaining the bacterial lysed cold along the process and add proteases inhibitors.
@Elizabeth Ferrer
Thanks for your help
Yes,My suspicion would be different ORF too.Do you think it is possible to express different protein according to one gene but in different colony?If one colony(colony 3) have a serious mutation in its exprssion system,it would produce a different sub-protein?
Or do you think it is possible the proteins of colony1/2 and 3 show a different mobility just because of their different tertiary structures produced by different bacterial expression system?And some tertiary structure is so stable that it can anti-DTT and SDS in SDS-PAGE?
Degradation is a possible explanation.But why did only colony 3 shows this degradation problem if this protein A prefer degradation?
In fact,this is not very important for me,but it is a interesting thing.Thanks for your recommendation,I will repeat this experiment and watch what will happen...
best regards
Hi Dali,
I would say it is an interesting scenario and just a random situation and there can be more than one factors like: contamination, lack of uptake of pET28 by that clone, reduced action of IPTG or there maybe other factors that suppressed the expression of the recombinant protein but enhanced the expression of another protein which we are seeing here in the 3rd clone.
Just repeat the experiment with the same conditions and hopefully you will not face the issue again. You can also pick some more clones and analyze them on SDS PAGE to confirm whether this is a rare chance or there are others like this too. Hope that you will get things right in the next run.
Best of luck :)
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