I am facing problem in protein expression from the gene cloned in pET28 vector (BL21 host) which was previously expressing happily. Now I am trying to induce protein from same construct in same host but it is not expressing. I have tried different concentration of IPTG induction but no expression. Even I freshly transformed plasmid in BL21 but still it is not expressing. Why is it happing like this? Kindly suggest me some solutions. Thanks in advance.
Hi, Sachin,
How long have you keep the construct? Did you check your protein whether it is toxic to the host cells.
if your protein of interest is toxic it may reduced the yield of the protein, or sometimes your protein of interest will not appear in the gel.
Sorry, I am answering your question based on my knowledge and experience, if there is something wrong please correct me.
Thank you.
This may be too obvious but:
1. Have you checked that you reach the same OD each time before induction?
2. What temperature are you using for expression?
3. How much IPTG are you using? Most sources say that anything over 0.25 mM is excessive though this probably won't affect expression. Of course too little IPTG will affect expression.
4. What growth medium are you using?
Hi all ... thanks for your answer and suggestions
Usually I give IPTG induction once culture reaches OD of 0.5 at 550 nm. I have tried 1mM IPTG induction but still it was not expressing expected protein. Further I tried 3 different concentration of IPTG as 1mM, 2mM and 3mM but still no results. Since the construct was previously expressing so there is no doubt about constuct preparation. I just revived culture from glycerol stock and used for protein expression with IPTG induction. After this I did fresh transformation, still no results. Once I will try to lower the concentration of IPTG.
Are you sure the gene cloned into pET28 is not mutated? Have you sequenced again the insert?
Jose
Hi Sachin,
Hope this helps...
Good luck... :)
Shridhar.
Hi Sachin,
I agree with the suggestions so far:
Check clone-if it was stored in a 'B' strain instead of a cloning strain there is the possibility of recombination. Re-prep it in a recA/endA strain and re-sequence.
Shridhar's suggestion that you could have used a non-DE3 host would also account for no expression as the T7 driven plasmids need the T7 polymerase produced from the DE3 integration. Check that you have DE3 strains.
If both of these are checked and OK then the protein may be toxic (but why not before?) then you can perhaps express it by the addition of 1%glucose (final) to all your transformation plates and expression media. This blocks leaky expression from the both the DE3 integrate and your plasmid and allows cells to grow as normal until the point of induction. Can make an all or nothing difference to protein expression.
Also re-check antibiotics and IPTG stocks and concentrations.
Are you using same temperature as before?-higher temperatures may have driven the protein into inclusion bodies and, depending on your extraction/detection method you may only be selecting 'soluble' proteins.
If you had expression of this construct before it has to be one of these things
Good Luck
Hi Sachin,
Is your protein expressed as soluble or insoluble protein?
You have to transformed the construct freshly..it is happen..I have attached a paper which will help you.
Bro have you find any solution with this problem? I am encountering the same problem as no expression after a high expression in same vector though all is well.
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