Hi there,

No there would be no difference at all as the plasmid is empty therefore there would be no new plasmid driven protein expression during induction. Is the protein you try to express meant to be tagged? If yes have a go with Western Blot on cell extracts using anti tag antibodies.

Many things can go wrong with protein expression. Can you describe your experiment a bit better? What vector are you using, what E.coli strain, what are your expression parameters? Dominique is right however, there should be basically no difference before/after induction, except maybe an ever so slight induction of T7 RNA polymerase running at 100 kDa.

Thanks for the feedback. I am using pET-28a+, host strain BL21(DE3) and Rosetta(DE3) pLysS. I have tried to induce the protein at OD 0.6 at 16, 25, 37 C at different IPTG concentration : 0,1, 0.5 and 1.0 but none seems to work. So my supervisor was suspecting that the induction has failed to work 

one more question why wouldnt empty pET give a 5-6kDa product starting from the initial met and ending with the stop codon?

Hi!

I agree that you could expect a ~ 6 kDa peptide since you have a start- and stop-codon with "something" in between (including His-Tag, T7-Tag etc.), but it might happen (especially with short peptides) that it becomes degraded by its host - or this is what is often assumed to cause bad expression for short sequences, this is why they are often expressed in fusion with other proteins. So the "empty" plasmid as a control maybe does not give you an answer whether your expression protocol is appropriate.

I agree with Dominique that Western Blot analysis against e.g. His-Tag would be the easiest way to see whether your protein of interest is expressed. In case you did not remove it for your cloning procedure....

Best, you take samples of your culture from e.g. hour to hour after induction and check it with western blot. From this, you can optimise your expression conditions if necessary.

Best regards,   Christian

You are right in principle that the MCS region is induced and for pET28a you might just about be able to see a 6 kDa band on a 16% or 18% SDS gel. In general however, many expression vectors have shorter MCS and are not resolved by standard 12-15% SDS PAGE. If you want to test induction I would therefore look for a proper positive control.

In my experience, the T7 system is quite robust. So, unless you have reason

to believe that your IPTG has gone off, I would tend to think that induction is not your problem.

Assuming that you confirmed the integrity of your construct by sequencing, I would think it more likely that you observe an intrinsic problem in the construct you try to express. Some proteins constructs can be very difficult to express in E.coli (not talking about solubility, but actual expression levels). These can include intrinsically unfolded proteins, multidomain proteins or proteins, which are usually part of a multi-protein complexes.

Are you expressing a full-length protein? Can you vary your construct boundaries? What about co-expression with a binding partner? Alternatively, you can try a different expression tag - a solubility tag like MBP, Thioredoxin, or GST can sometimes also help expression levels.

Hi Markus and Christian ,

Thank you for your explanation. 

Markus, I do have some truncated construct with insert (consisting of the motif) of ard 500bp and 261bp. Do you think if they are too small to work with? (My full length insert is ard 700). I need to complete my project in less than two weeks time. I dont have much time to play ard. But I will definitely include that in my future work 

Hi Shanice!

Since it urges, I would like to give you some infomation about a protein expression I had tried earlier.

I expressed a 15 kDa protein. I never saw a protein band after induction, but found it by western blot. After optimising expression (LB medium, growth of culture to OD600 ~ 3-4) I recovered around 4 mg protein per liter of culture (with Rosetta cells without pLysS) - still without seing the protein after coomassie stain of the total protein fraction, but it became visible after nickel affinity purification. So depending on how much protein you actually need, maybe it is already sufficient... Western blot analysis would still be great to identify the protein - although failure of induction, as Markus mentioned, is unlikely.

To note, I extracted this protein from total cell protein since it did not need to be folded within the bacteria and was mainly in the insoluble fraction. I cannot estimate losses if you have to isolate your protein from the soluble bacterial protein.

thank you Christ. Really appreciate your feed backs and tips here.

I will try conduct a western blot tomorrow and hopefully can get some luck !