protein production didn't work! - (Sep/12/2006 )
Hello
I am trying to produce a viral protein (a protease of 17 kD) in fusion with the GST. I am using the PGEX-KT plasmid and the BL21 pLysS bacteria. I've applied two different conditions of production; 0,1 mM IPTG at room temperature and 1 mM IPTG at 37 C°. after 3h of incubation, no protein production was detected on the SDS-PAGE though sequencing has confirmed the presence of the insert in the plasmid (induction at D.O=06). any one has encountered such a problem ? thanks for help.
Here is something simple
try dropping the induction temperature, try 16 Celsius. (Compensate by increase induction time)
I don't have my books with me right now, but you can certainly increase the amount of IPTG.
Thanks for your reply,,,
do you mean induction at 16 C° with 1 mM IPTG?
do you think that the presence of rare codons in my gene can inhibit the protein production? I think it may have an effect but can this abolish the production completely ??
are you looking at the correct size for your protein? (just a thought)
even if rare codons are present you would still expect to see a small amount of protein expressed though this may not be easily visible by SDS-PAGE.
you could carry out a western blot and probe with an anti-gst antibody which would tell you if any protein is being produced. even better to do a western with an antibody to the expressed protein if you have one.
alternatively you could attempt a small-ish scale purfication on some gst-resin and see what you get...
hope this helps
enjoy
do you mean induction at 16 C° with 1 mM IPTG?
Ummm... no, not really. I mean you could induce your cells at 16°C. The amount of IPTG can vary from 0.1mM up to 2mM. Personally, for no reason at all, I would start at 0.5mM IPTG and see where that goes. Normal induction time can be 3-5hr, so I usually induce over night when at 16°C.
Yes, it will have a very severe effect if lots of protein is desired, more so if mutiple and importantly the same rare codon appears several times in the protein. As for abolished production, well it won't be zero, but I won't pin my hopes on see it on as a band on a gel. Western blot would probably show it up, but that isn't why the IPTG induction was done right. Try Rossetta cells... they have a revempt tRNA system. So rare codon shouldn't be a problem.
Anyway, if IPTG don't give you enough protein, and you are using something like Rossetta, maybe you should try a different induction system, GST, His-tagged, MBP, protein A, and intein-CBP.
If it is none sugar based induction system, you can used terrific broth to get more cells.
However there is a worrying point which hangs around, is your protein toxic? If it is, the majority of the cells would lose the plamid and produce very little of protein. Did you sequence a several isolated single colonies? PCR is fantastically powerful. It it comes to it, you may have to move host species.
just a thought, did you design your vector yourself. if so are you sure everything is in frame?
Kathy: yes i designed my vector myself but sequencing has confirmed that everything is ok and in frame ( I sequenced a one single colony, no more).
Perneseblue: yes the protein is known to be toxic for eukaryotic cells, probably it may have the same toxicity on prokaryots too!!! If so how can I overcome this problem ???
Yousef
if it's a problem of toxicity , the bacteria would die after induction. You should monitor the OD of the bacterial culture after induction. If OD doesn't decrease, your protein is not toxic.
If your protein is toxic, you can try to reduce the induction time to 1h. you should do a time titration and a IPTG concentration titration.
With toxic proteins, it can be possible that the expression is not strong enough to visualize it on SDS-PAGE. you should do a western-blot.
Yousef
Is there anything you can do to reduce the the toxicity of the protein? Somekind of inhibitory molecule? Probably not. What is the mode of toxicity?
Well you would probably have to grow lots of cells. Having the protein excreted from the cell might help. Though I don't know if that is applicable or possible to your protein. Furthermore excretion required a little modification, so it is fusion protein rather then the native protein.
Well, like missele suggested, do a small experiment and see if the protein is toxic to bacteria. If it is, well you have to play a balancing act, express the protein right to point before the OD falls. Stop at that edge, kills the cells and extract the protein. And as mentioned earlier grow lots of cells.
A random thought,could try playing with archea expression systems. Could an archea be resistent to the toxic effects? What do you know about this protein. Where does it come from originally. the cell that produces it should be resistent to the protein's toxic effects... unless the protein in question is synthetic..... what is the mode of toxicity.
thanks for your remarks,,
in fact this protein is a protease of a viral origine ; the 2A protease of ECHOvirus6,,, it acts by cleaving cellular proteins and most importantly the eukaryotic translation initiation factor eIF4g. it is though responsible of the cellular shut-off phenomenon.
according to your suggestions I'll try to produce this protein in a Rossetta cells at 16°C overnight with D.O surveillance at least for the first 2- hours after induction,,,i hope this may help resolving the problem,,,
do you know if repeated cycles of thawing/ freezing can reduce the IPTG activity? can this be the cause of the problem as i used to use the same IPTG stock for inductions? I can imagin that the more fresh is IPTG the better it is but can an old stock be out of activity ?