Hi Chandrima,

are you using IPTG or auto-induction additive to promote expression of your protein during induction?

1% Glucose added to Terrific Broth is not a normal induction method, so I would suggest trying addition of 1mM IPTG for 1-3 hours at 37˚C as a worthwhile test.

Please feel free to discuss this here further, especially as you test different conditions. Good luck!

Hi Robin,

I am using IPTG for expression. I am optimizing IPTG concentrations from 0.05 onwards and kept for 2 hrs at 37C. None is showing any expression.

Can I ask why you've added glucose? This is normally used as a suppressant for leaky expression from Lac operon-driven genes so it may be countering the IPTG, especially at low levels. Also - have you tried IPTG concentrations of 0.5 or 1mM final in the inductions?

Secondly - the obvious questions. Have you ensured that your reading frame is cloned in frame, that there are no spurious stop codons, and that the codons are not obviously troublesome as far as expression is concerned?

exactly right! I would suggest you the same as of Robin Dickinson.

Also consider that either your protein is extracellular or not.

you can also increase the time duration.

Add some protease inhibitors while checking expression.

Hi Chandrima,

You asked a very general question and I guess many of us have already had a similar problem. It might be a silly question, but how do you know there was no expression of your protein? Do you just judge from SDS-PAGE, do you have antibodies you can use or some kind of specific activity you could measure? It could be that you are just expecting an overexpressed protein which is not necessarily the case even with T7 promoter. Also, did you check for eventual degradation products (lower molecular weights) that might appear with time (not really expected in 2 h induction, though)? You could change the expression strain (to e.g. +pLysS) or change the medium, which is the easiest thing to do. I would go to a minimal medium with glucose and then prolong the expression to at least 4 h, taking samples at hourly intervals for analysis. Optimization of IPTG concentration is not your first priority, for your initial experiments you should take 0.5 mM (as Robin suggested) and only when you get detectable expression go for lowering to whatever is needed. In principle, lac promoter (for T7 RNA-polymerase expression) is not dose-dependent at the level of a single cell and a very low concentration is needed to actually induce expression. And, as Robin already mentioned, do check the DNA sequence for mutation or eventual frameshift behind the 5' tags.

Good luck,

Marko

Hi, Chandrima: I do not write English very well (I hope to be understandable)

I also express recombinant protein. I use the pET-Topo vector and strain BL21 (DE3). I do not use glucose. My reading frame is correct.

The expression of time was 2-4 hours at 37 ° C and 18 hours at 28 ° C. Did you try these variables? . I still can not get my recombinant protein. I still can not find the error.

I will use the BL21 STAR strain, I have read bibliography and it has a greater expression efficiency. Any question I am at your disposal.

Hi Vanina,

I have finished the new set of experiment with LB media for 16 hours with and without glucose. I will let you know once i get the result. i have not tried with low temperature, but will surely try.

Please feel free to suggest if you get have anyother suggestion

Hi,

In some cases, when you wnt to express heterologous proteins in BL21 DE3 strain, you may use BL21-CodonPlus (DE3)-RIL. As mentionned in the Stratagene booklet (page 3) "Efficient production of heterologous proteins in E. coli is frequently limited by the rarity of certain tRNAs that are abundant in the organisms from which the heterologous proteins are derived. Forced high-level expression of heterologous proteins can deplete the pool of rare tRNAs and stall translation. BL21-CodonPlus strains are engineered to contain extra copies of genes that encode the tRNAs that most frequently limit translation of heterologous proteins in E. coli (https://www.agilent.com/cs/library/usermanuals/public/230240.pdf).

Maybe you are in this situation, so try to change the bacterial strain.

Good luck!

Hi,

I would try other DE3 strains. I have found that expression of some genes is very dependent on the E.coli strain used. Also, the added glucose should not be a problem; I routinely add a low concentration (3-5 mM) of glucose to my expression media. It helps prevent any expression of the gene, which may be detrimental to growth, before induction with IPTG. IPTG induction is very strong and overpowers any glucose catabolite repression of the lac promoter controlling T7 RNA polymerase transcription.

I would also try Studier's auto-induction media. Once you make up the solutions, growing the E.coli is easy, as there is no monitoring the growth. It has worked very well for some, but not all, of my proteins.