Hi.  Assuming that your gene is appropriately cloned into the vectors and you are using BL21(DE3) or a comparable strain that contains the T7 polymerase there are a number of possibilities.  First and foremost you need to ensure that your plasmid is stable.  There are a number of common mistakes that can be dealt with easily.  If you use rich media with yeast extract you can have leaky expression and resulting toxicity.  Most common sources of this contain a bit of lactose or a similar compound.  This can easily be dealt with by adding glucose to your culture (suppressor of lactose), or, a better solution is to use MDG media.  This media was developed by Bill Studier at BNL and is extremely repressing.  See attached document.  We use MDG plates anytime we are transforming BL21(DE3) cells as well as for all of the overnight cultures in the expression host.  If you plate the transformation reaction on MDG + antibiotic plates (pET28 is Kan resistance correct) and then grow single colonies from this in MDG you will have, as best as is possible with that vector, good starter culture.  The next step is to make a glycerol stock.  A common problem here is using a 20-25% Glycerol stock.  At this concentration every time you go into the stock some of the cells at the top thaw and then refreeze.  In my hands, and others, this enriches for cells that have altered or damaged plasmids (in the case of Amp resistance it enriches for cells that lost plasmid and since amp is hydrolyzed and bacteriostatic you will often see the -Amp containing cells overgrow the appropriate cells during large scale culture growth).  Instead, its better to use an 8% glycerol stock.  This minimizes any thawing and refreezing and we see little if any difference in survival.  For a really toxic gene you should probably test to see how well you are doing with plasmid maintenance.  The simple test is serial dilution of your culture onto plates with Antibiotic, no antibiotic, IPTG, and IPTG plus antibiotic.  From this you can determine the number of cells, cells with plasmid and cells with plasmid able to express your protein. 

  Finally, I strongly urge you to try one of the autoinduction media.  These are very repressing and, if you have a good glycerol stock, the most likely to yield expressed protein.  There is no media or protocol that guarantees expression every time, but this is as close as it gets.

  In addition, you can try several other variations for example using a plasmid with rare tRNAs such a pRIL. Reducing growth temperature (typically though if it does not express at 37 its less likely to be expressed at 30, 27, 20, or 18C, the caveat though is it might be being degraded at 37 and stabilized at lower temperatures), Check to make sure that there is little RNA structure near the 5' end (we have had luck by increasing the AT content near this end, although there is some debate whether this actually is generally applicable, and most people are working with an N-terminally tagged protein so its less critical).  For some really toxic genes we have used an Arabinose inducible T7 based system. 

  Things that rarely work are lowering the IPTG concentration.  As has been shown previously, IPTG works more like an on/off switch. By lowering the IPTG you tend to alter the number of induced cells (stochastic) rather than lowering the rate of synthesis in every cell.  John Dunn spent several years trying to modulate the IPTG uptake to no avail.  The caveat to this statement is that your GOI may have an unusual effect upon bacterial metabolism and by some curious chance allows a more graded IPTG response.  For example, the earliest membrane proteins to very highly overexpressed in E. coli turned on the lipid synthesis pathway which resulted in large amount of intracellular membranes containing the expressed protein.  However, many membrane proteins are now reasonably well expressed in E. coli but it required optimizing media (autoinduction media works remarkably well) and minimizing toxicity.

is your strain BL21 or BL21(DE3)?

In addtion to asked by Shridhar, check if your ORF is in frame with the start codon of pET vector.

dear Imran,

first check if the gene of interest has been cloned in way it has reading frame properly. to do this you can isolate recombinant pET vector and design a primer flanking to gene of insert. if fragement is too large then use to primer forward and reverse both. do sequencing of inserted fragement using these primers.  once you get the sequence, align them with vector and find if any unusual modification if occured during  cloning.

good luck

Here are several possibiltes for your problems,

1, just like Alfonso and Kumar suggestion, make sure that your interest gene was  successfully ligated in pET system;

2, even T7 promotor is a strong promotor in pET system,  they offen need induction of IPTG (0.1-1 mm) for enhanching  the expression of your target gene,

3, some culture condition and medium also somewhat affecting the expression (try, LB,Terrifc broth or autoinduction medium in several diffent temp culture),

4. if the expression still not satisfied enough, may you need to change the plasmid system...

success for your project

Dear Imran,

 I was also experienced same problem while i'm trying to clone and express human vegf with pET system but later it works fine .

In your experiments i didn't  understand in which strain   your gene was cloned ?  because for my experience DH 5 Alpha is best for the cloning  and BL-21 is good for expression. first thing  you need  concentrate much during  transformation step. Second as Mr.Asep said you have to  optimize with IPTG concentration (Dosages v/s temperature). Third one try to maintain positive control and negative control for each step including LB broth , competitive cells efficiency  and etc . finally  I think after cloning you can use restriction enzymes followed by electrophoresis to confirm . All the best

I suggest you first that You can check with the restriction enzyme for the product and also an enzyme with restriction site inside the cloned product (to reconfirm if the product is right). Check the reading frame of the gene before cloning.  I have used pET  vector and they are reliable. 

Check for the expression with other strains like C41 etc and I suggest you to play with IPTG concentraion. Try inducing with low concentration of IPTG and induce overnight (8-10hrs) at 16 degrees.

Induce at a lower O.D of bacterial growth 0.4-0.5. Good luck 

Hi.  Assuming that your gene is appropriately cloned into the vectors and you are using BL21(DE3) or a comparable strain that contains the T7 polymerase there are a number of possibilities.  First and foremost you need to ensure that your plasmid is stable.  There are a number of common mistakes that can be dealt with easily.  If you use rich media with yeast extract you can have leaky expression and resulting toxicity.  Most common sources of this contain a bit of lactose or a similar compound.  This can easily be dealt with by adding glucose to your culture (suppressor of lactose), or, a better solution is to use MDG media.  This media was developed by Bill Studier at BNL and is extremely repressing.  See attached document.  We use MDG plates anytime we are transforming BL21(DE3) cells as well as for all of the overnight cultures in the expression host.  If you plate the transformation reaction on MDG + antibiotic plates (pET28 is Kan resistance correct) and then grow single colonies from this in MDG you will have, as best as is possible with that vector, good starter culture.  The next step is to make a glycerol stock.  A common problem here is using a 20-25% Glycerol stock.  At this concentration every time you go into the stock some of the cells at the top thaw and then refreeze.  In my hands, and others, this enriches for cells that have altered or damaged plasmids (in the case of Amp resistance it enriches for cells that lost plasmid and since amp is hydrolyzed and bacteriostatic you will often see the -Amp containing cells overgrow the appropriate cells during large scale culture growth).  Instead, its better to use an 8% glycerol stock.  This minimizes any thawing and refreezing and we see little if any difference in survival.  For a really toxic gene you should probably test to see how well you are doing with plasmid maintenance.  The simple test is serial dilution of your culture onto plates with Antibiotic, no antibiotic, IPTG, and IPTG plus antibiotic.  From this you can determine the number of cells, cells with plasmid and cells with plasmid able to express your protein. 

  Finally, I strongly urge you to try one of the autoinduction media.  These are very repressing and, if you have a good glycerol stock, the most likely to yield expressed protein.  There is no media or protocol that guarantees expression every time, but this is as close as it gets.

  In addition, you can try several other variations for example using a plasmid with rare tRNAs such a pRIL. Reducing growth temperature (typically though if it does not express at 37 its less likely to be expressed at 30, 27, 20, or 18C, the caveat though is it might be being degraded at 37 and stabilized at lower temperatures), Check to make sure that there is little RNA structure near the 5' end (we have had luck by increasing the AT content near this end, although there is some debate whether this actually is generally applicable, and most people are working with an N-terminally tagged protein so its less critical).  For some really toxic genes we have used an Arabinose inducible T7 based system. 

  Things that rarely work are lowering the IPTG concentration.  As has been shown previously, IPTG works more like an on/off switch. By lowering the IPTG you tend to alter the number of induced cells (stochastic) rather than lowering the rate of synthesis in every cell.  John Dunn spent several years trying to modulate the IPTG uptake to no avail.  The caveat to this statement is that your GOI may have an unusual effect upon bacterial metabolism and by some curious chance allows a more graded IPTG response.  For example, the earliest membrane proteins to very highly overexpressed in E. coli turned on the lipid synthesis pathway which resulted in large amount of intracellular membranes containing the expressed protein.  However, many membrane proteins are now reasonably well expressed in E. coli but it required optimizing media (autoinduction media works remarkably well) and minimizing toxicity.