I am trying to purify a protein from a thermophilic bacteria (gene cloned in DH5 alpha) but I am not getting expression of my protein after IPTG induction. I have tried with different expression vectors of E.coli, different incubation time, diff. conc. of IPTG (up to 1mM) but still it was not coming.
always worth trying auto induction. Its simple and very easy
http://www.bioc.aecom.yu.edu/vetting/PROTOCOLS/noniptg.html
DH5 alpha is not suitable for IPTG inductions, you should use BL21 (DE3) for example. Rosetta and CD43(DE3) will also work.
IPTG does not directly induce expression from the plasmid, but it triggers the expression of T7 RNA polymerase on the chromosome of some strains (those bearing the "DE3" insertion). This T7 polymerase will recognize its promoter on your pET plasmid, and induce protein expression.
@ James: Autoinduction won't work in DH5 alpha (no lacY transporter).
@ Suraj: I doubt your problems have anything to do with the origin of the gene (Taq DNA pol is from a termophilic bacteria, and yet it is expressed quite well in E. coli. Same goes for Pfu, Pwo, etc). You haven't said what vector you're using to express your protein, but if it employs a T7 promoter, then you should not get expression in DH5 alpha (it does not express T7 RNA pol). You need a lambda DE3 lysogen for that.
@alejandro: the gene is in pET11a vector and i have tried with different expression vectors of E.coli.
@rioux: i have treid with BL21 DE3, rosetta and c$#, but still the protein wasn't expressed.
@James: is there anything we can do with the media i am using like adding glycerol or glucose or sorbitol could make a difference or not.????
and thanx to everyone for your responses
OK, if it's pET11a then you definitely will not get expression in DH5 alpha. Use BL21(DE3) instead.
There are a number of checks you can run to pinpoint the source(s) of your trouble:
1- Have you tried with another construction, known to express well? This is necessary to ensure that your culture conditions and host strain are OK.
2- Have you verified the sequence of your construct? Is it in frame? I've never worked with pET11a, but in pET11d (the only difference being the reading frame, if I recall) you have to use the ATG provided by the NcoI site, and have to include your own stop codon.
3- How do you know there is no expression? It is not the same to have low expression (detectable, e.g., by Western blot and indicative of poor translational initiation, degradation, etc) than no expression at all (which suggests that there is something wrong with the construction itself).
Hope it helps
Try following the protocol in the link very simple as mentioned requires something like bl21 and an amp resistant plasmid. Also as suggested check your construct do you have a requirement for a co-factor or do you have something like a transmembrane domain which may screw things up. Alternatively try a fusion such as MBP although this can drag through poor quality protein. Try orthologues of the gene or switch expression system to something like bevs or yeast. But as previously suggested how have you checked for expression? Have you looked the pellet (insoluble fraction)
James
@alejandro martin: yeah, the sequence is ok but your third point seems valid as I got very less expression of my protein in C43(DE3) but the expression was also present in uninduced culture so I am a bit doubted it that is my protein only.
@james: thanx for the protocol sir, I will surely try that and will inform you. I read that, but i think my protein is toxic as after few hrs of induction the cells were dyeing so i think BL21 wont work in the case.....and i checked the pellet too, bt no positive results...
Be VERY careful when working with toxic proteins, as you will inevitably select faster-growing clones which more often than not simply do not express your target protein. Some guidelines:
- Make a prep of your clone and send it to a sequencing facility. If everything is OK, use that particular prep for transformations, etc.. Avoid using minipreps from expression or serially passaged cultures, which may have accumulated expression-impairing mutations.
- If possible, switch to a more modern vector (say, pET28a+). In pET11a there is some transcriptional read-through from the AmpR gene. If the protein is really toxic, you should see toxicity even when the plasmid is propagated in cloning strains.
- Do not, ever, store your recombinant strain in Petri dishes in the fridge. Transform the plasmid in your desired host, and the next morning grow small scale cultures and prepare glycerol stocks (don't let the cultures reach stationary phase either). Aliquot them in small portions, and thaw/discard an aliquot every time you start a new culture.
- Never do serial passages in Petri dishes.
- Always include glucose in your cultures/plates whenever you do not intend to express the protein (i.e. when propagating your clones or making stocks). From 0.2% to 2%, it doesn't matter the exact concentration. This will repress the lacUV5 promoter driving T7 RNA pol in the host by inducer exclusion.
- Try the pLysS variants of the DE3 lysogens you're using. This will minimize basal expression as well.
- You really need a positive control (a well-behaved protein cloned in the same vector). That is an essential sanity check under your circumstances.
Hope it helps
@alejandro martin thank you vey much for your valuable suggestions. Will try to follow the instructions to a maximum extent (already following some of them) and hope this will help me.
Switch to a host that has a really tight control of T7 polymerase expression (i.e. not lac-dependent). BL21 AI (arabinose-inducible) from Invitrogen is better for that than BL21(pLysS). Be sure to induce with L-arabinose, not D, and add also IPTG if your expression plasmid carries a lac operator. Also, check the sequence for rare codons, such as AGG/AGA, CTA etc. If they are frequent, transform your host with a plasmid providing the corresponding tRNAs, such as pCodonPlus or pRARE.
thanx @beguin sir. and optimal concn. of glucose and glycerol in the LB media also worked... thanx to all of you..
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology