I have previously cloned, induced and standardized the method of purification of a membrane protein of 3kb but couldn't store it till now. I am now trying to induce the protein again using the same plasmid (pET21b+gene of desire) transformed in E.coli BL21 but I can't get induction in the same conditions as previously.
I have tried to double digest the plasmid containing the gene of desire with the restriction enzymes BamHI and XhoI by which the vector and the gene was double digested before ligation. The total plasmid (pET21b+gene of desire) was 8kb. The plasmid was not digested, therefore I tried to digest the plasmid from glycerol stock (pET21b+gene of desire in DH5a). The plasmid was also not digested. I tried with the glycerol stock of previously induced protein (pET21b+gene of desire in BL21). The digestion was successful and the bands of 3kb(gene)and 5.4kb(pET21b) was obtained but I couldn't get induction. I isolated the plasmid and transformed in fresh competent cell, but still couldn't get induction.
I was inducing the full length protein of 99kDa at 298K for 8hrs at 2mM IPTG. Previously I have tried to induce it at 0.5-1mM IPTG but can't induce.
In literature the truncated form of this protein was induced at 2mM IPTG 303K for 4hrs.
In my case the induction was not much increased at 298K for 8hrs. The protein is a e.coli protein. Please help in troubleshooting.
Question
I can't get induction of a previously induced protein using same induction procedure
All Answers (16)
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I think you almost mentioned this (but not quite), but did you plate out bacteria from the glycerol stock that you know worked, pick a few colonies and grown liquid cultures to induce from there?? -
Hi Papri
It is seen very often that the insert cloned is not very stable (or is not tolerated) and the recombinant clone lose it over a period of time...or may accumulate variation that renders it inactive (as far as expression is concerned)...even when stored in perfect conditions.
In such a scenario if you sequence the plasmid from the clones which have given you protein previously (or digested as expected) and are not working now...you may find that the insert sequence may have been deleted or mutations may have accumulated in the ORF and henec you don't see induction.
As suggested by Stephen above..you pick more colonies and check for induction.......
One can also express such proteins in a low copy no plamid so that the intracellular levels of recombinant protein would be low and will not interfere with the general metabolism and may be tolerated in the cell
Ajay -
Hai friend
i think the problem is with your clone because after a long time storage in glycerol some of the clone loss its viability. so please check the DNA seq of your clone and check with the EXPASY translate. if the problem is not with the clone please make sure of the E.coli cells and IPTG. BEST OF LUCK FOR YOUR RESEARCH. -
We often see that an expression strain that has been stored can lose it's ability to express the desired protein. However, if a fresh colony of DH5a with the correct plasmid was stored away you SHOULD be able to make a mini prep from that and transform to BL21(DE3) or similar and be up and running again.
That you are having trouble digesting the DNA you have stored away does make me suspect that you have some plasmid stability issues. I would pick several colonies from the DH5a strain (streak for singles first, or course!) and sequence them (at least over the cloning junctions).
If the plasmid looks good, transform into fresh BL21(DE3). Follow your previous protocol, but include (if possible) a positive control - a gene you know expresses reliably. This will confirm your IPTG hasn't gone off or something weird.
If you still see no expression, then how are you judging expression? Coomassie staining, activity, or western blot? What level did you get before, and what is your limit of detection? Have you checked the pellet to see if the protein is being expressed, but is insoluble? Some proteins are very sensitive to the exact conditions (Temp, OD at time of induction, media composition etc), and if you are off by a bit, it can tip the scales in the wrong direction. If it is insoluble, try 16C instead of 25C. -
Richard has covered this beautifully. I'd say tackle one problem at a time. First focus on recreating a glycerol stock in DH5a to use as a plasmid source. Streak the cells that have gone bad and raid your freezer for leftover plasmid DNA aliquots (any tiny amount, or maybe your mini-preps from when you first made it?). And sequence, definitely sequence. Only when you are sure about your DNA go and address the expression issue (and do use a fresh transformation in BL21 for expression).
A point not mentioned is to make sure that the appropriate antibiotics have been used - sometimes it could be something as trivial as forgeting to add the selection antibiotic when you grew your DH5a stock overnight. -
also you should keep in mind that T7 polymerase expression might vary with time..see the paper below for the details:
Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545050/
because of this, i personally prefer to make a new transformation every time - and use the same plate for 1 week only
best of luck! -
@Iryana
Thanks Iryana for an informative paper on this problem
Ajay -
Dear Friend,
Although the subject has been covered very well but still Icontribute something. First induction membrane proteins is very difficult simultaneously ur cells r loosing plasmid. Two best strategies u can apply is make fresh transformation each time and increase antibiotic pressure. So, the plasmid is always under pressure. Secondly, increasing inducer concentration and time doesnt mean highest expression. For that u have do basic induction concentration experiment for proper induction. If u can specify which type of membrane proteins are u dealing, I might help u. I am also working on transmembrane domain proteins. I did induction but hardly it was visible by Coomassie staining. So, I had to do Western blot for confirmation. pET series has 6x His tags which can be easily deteced by Western blot. -
Dear all,
Everything has been said regarding the biology. I found that by using glass erlens it may compromise your protein production... because if you treat them with bleach, it "sticks" to the glass (porous material) and get release during your culture (does not affect DNA prep). do not let bleach to long in your erlen. I personnally switch to plastic erlerns for tissue culture tnat I was washing myself to reuse them (no bleach). With time they tend to get not as good as at the beginning.
Anyway, my feeling is you do not have 1 problem, but many small problems that accumulate and at the end you have lost your production.
For production in E. coli, I ALWAYS make a new transformation and ALWAYS take all the clones and use them for the culture.
Good luck.
Patrick. -
Dear
As mentioned in most comments pET-gene combinations may be very unstable and loose the ability to express the cloned gene often as a result of rearrangements in the plasmid that effect the expression unit. This is particularly the case when uncontroled expression a protein that is toxic to E. coli, as may very likely be the case with a membrane protein. The reason is leaky expression of the T7 RNA polymerase under uninduced conditions.
A possible solution is suggested in the following 2 papers:
Mertens, N, Remaut, E, Fiers, W. Tight transcriptional control mechanism ensures stable high-level expression from T7 promoter-based expression plasmids. Bio/Technology 13, 175-179, 1995.
Mertens, N, Remaut, E, Fiers, W. Versatile, multi-featured plasmids for high-level expression of heterologous genes in Escherichia coli: Overproduction of human and murine cytokines. Gene 164, 9-15, 1995. -
Dear
We have some experiences with the pET series vectors and it sometimes show very in consistance in restriction digestion after storage in glycerol. You can check for the insert in your plasmid by PCR specific to your gene.
It is observed that over a period of time, the T7 based E. coli vectors loss its transcriptional ability. This may be because of mutations in promoter region or transcription regulators. It is advisable to sub clone your gene into your vector of interest again which may solve all your problems related with protein expression.
All the best. -
Papri,
I'm trying to work out if you have done this already by reading your first message. If the recombinant plasmid you have transformed into E.coli has stability issue. What about the isolated plasmid you originally generated after you've done the sequencing but before you did the transformation? Would you still have a plasmid prep stock of the original verified DNA stored at -20 C? It is less likely for anything to happen to the frozen DNA kept in the freezer.I often have both transformed stock of cells as well as the pre-transformed plasmid DNA frozen for storage.
Best wishes -
I had similar problems but i was dealing wit yeast proteins expressed in Ecoli. I could overcome this by changing the temperature of induction. I did everything at 37 degrees.
May be you could alter temperatures and check.
Also, have you checked if your protein is soluble? may be it gets induced and could be lost in the pellet?! -
Thanks to all of you for looking into the problem and trying to finding its solution.
I have already tried to express by culturing the cell from previously induced glycerol stocks of BL21 but can't get the result. I have checked for IPTG and the marker Ampicillin, they are all in proper condition.The trouble is nither in digestion nor in storing the glycerol stock.I have plasmid from many colonies of DH5a and transformed in BL21 but can't get induction.I used the same protocol as I got induction previously but can't get the induction band on gel. -
If you can't digest your insert out of the vector, it is very likely your plasmid isn't right. If the recombinant plasmid isn't correct, i would say there is no point spending efforts to get induction from this plasmid. I would go back to re-ligate the digested PCR insert into the pET vector again, sequence to confirm it (make sure that it is in frame) and keep this plasmids in the -20 freezer as a master copy. From there, at least you know the construct is correct, hopefully you will get expression again.
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I have got the induction by transforming my old plasmid in a fresh BL21 DH3.The only thing that I have change is the source from where I used to get the distilled water.The distilled water system which I used previously by me was out of order and has been repaired recently.
I am really thankful to you all as you have spend time to look into the matter and helping in finding out the cause of not getting induction.
Thanks and regards,
Papri
Popular Answers
Decreased gene expression from T7 promoters may be due to impaired production of active T7 RNA polymerase
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC545050/
because of this, i personally prefer to make a new transformation every time - and use the same plate for 1 week only
best of luck!