1) Did you check the protein expression on SDS-PAGE at time zero (without IPTG), then cell sample at time of induction (IPTG addition), then cell sample after several hours of induction on SDS-PAGE?

2) Also please check in your sequencing data if your gene of interest is in the right frame. Did you confirm the sequencing data by sending away your expression plasmid pet28a containing the gene of your interest?

3)Try and use BL21(DE3)pLysS E. coli cell strain as T7 Lysozyme encoded in a pLysS plasmid reduces the basal level of T7 RNA polymerase expression. T7 expression method is a system used for high-level protein expression and some amounts of basal expression of protein will take place in uninduced cells. If your protein of interest happens to be toxic to E.coli then in that case it is necessary to decrease the basal level of expression by using the above mentioned strain.

4) After transforming your expression plasmid in E.coli, what do the colonies look like? are they all the same size or some are big and some small? I am asking this because difference in colony sizes may be indicative of protein being toxic to E.coli

5) You can also use a low copy, T7 driven expression vector.

Hope all of the above is helpful in resolving your problem.

how you are checking your protein expression??? SDS-PAGE or Assay??? As Bala said first Check for Rare Codon..and Check your protein Size..and Transform your pET+Gene into Expression Host of E.coli..

BL21DE3 is pET expresion host try into it..and Induce your protein..with IPTG..

Try induction at less OD. About 0.4 @ 600nm then add IPTG for induction. check the fusion site by sequencing to confirm that.it is fused in frame.

I would suggest you to check if the protein is soluble or not. Sometimes, your recombinant protein may be toxic to bacterial cells and this will result in very low or no yield.

use BL21 Codon plus cells for protein expression

you may check the fram of your inserted gene, I mean is it ligated in invert position or

in correct orientation? if you have used same restriction enzyme to cut the vector and you gene then the chances for invertion are there.

After taking into consideration the above suggestions you can try low temperature expression conditions. Insoluble proteins have a better expression at low temperatures. Also check the quality of IPTG. Ultra pure IPTG is the best

If there are some rare codons in your gene, you can try Rosetta competent cell. it increases the expression of eukaryotic proteins that contain codons.

1) Did you check the protein expression on SDS-PAGE at time zero (without IPTG), then cell sample at time of induction (IPTG addition), then cell sample after several hours of induction on SDS-PAGE?

2) Also please check in your sequencing data if your gene of interest is in the right frame. Did you confirm the sequencing data by sending away your expression plasmid pet28a containing the gene of your interest?

3)Try and use BL21(DE3)pLysS E. coli cell strain as T7 Lysozyme encoded in a pLysS plasmid reduces the basal level of T7 RNA polymerase expression. T7 expression method is a system used for high-level protein expression and some amounts of basal expression of protein will take place in uninduced cells. If your protein of interest happens to be toxic to E.coli then in that case it is necessary to decrease the basal level of expression by using the above mentioned strain.

4) After transforming your expression plasmid in E.coli, what do the colonies look like? are they all the same size or some are big and some small? I am asking this because difference in colony sizes may be indicative of protein being toxic to E.coli

5) You can also use a low copy, T7 driven expression vector.

Hope all of the above is helpful in resolving your problem.

You can standardize the OD before the IPTG induction between 0,4 and 1,0 and evaluate the total protein crude extract.

For running the electrophoresis you should use a normalized volume, for example 0.1 DO of the original culture in order to compare the differences of the expression in time and in comparison with the control without IPTG.

In addition you can check the localization of the protein extracting periplasmatic, soluble cytoplasmatic, and insoluble cytoplasmatic proteins (the protocols are in the Novagen Manual available on-line).

thank for your all suggestion. i already try for many times with this gene. now i cloned another gene with another plasmid (pET 32a), with same method its work well. my gene expressed strongly just like in the teory. :)

in my oppinion, my preverious gene mybe toxic for e.coli, so it cannot be expressed. i also have been checked for the rare codon. both of my new gene and the old one have very simmilar codon profile. so i guest this is because the protein product of my old gene is toxic.

thanks for your suggestion.

If your protein is in frame, correct orientation and toxic for the bacteria, you will see that transformed strain should grow slower than the empty vector transformed strain (liquid culture) and that will also say you that your expression is leaky. IF the protein is toxic, a) you should switch to a better controled expression heterologous system (maybe BL21 or M15 cells), b) you can induce the cells with IPTG at OD=2 (worked once for me)

induce IPTG at OD=2? wow i never think about that. thanks for your suggestion. i'll try it.

If you're afraid your gene is toxic, you can always add a catabolic suppressor (glucose) to the medium to cut out any basal T7 RNA Pol production (and therefore gene expression). If you do add glucose, add it from a filter sterilized solution to your cooled media after it's been autoclaved as glucose can brown (Maillard Reaction) under autoclave conditions.

Take a look through the 11th edition of the pET system manual, it has a lot of great suggestions. I think most projects using pET plasmids can have most questions answered some where in there.

http://kirschner.med.harvard.edu/files/protocols/Novagen_petsystem.pdf

You can also use induction with lactose not IPTG. You put bacteria on minimal medium with glucose, they will grow slowly up to OD=0.2-0.4 with only one source of sugar, then wash them twice and switch on to minimal medium with lactose only. For the first 2 hours they will starve in protest then they will start produce the protein (I usually waited additional 4 hours). Protein will be produced in lower amounts so it could be much less toxic. However, You can have the similar problem as I had a long time ago: the protein was produced so quickly in such high amounts (using pET16b plasmid) that it has formed inclusion bodies, which I was able to partially solubilize only with 8M urea, SDS was not strong enough even for partial solubilization. The slow induction with lactose solved this problem.

Auto-induction medium like those detailed in Protein production by auto-induction in high-density shaking cultures (Dr. William Studier) found here (http://wolfson.huji.ac.il/expression/local/bacteria/studierAutoInduced.pdf) have given me more success in gradual T7 Pol production and therefore protein production.

I can also report that the auto-induction medium from Studier (2005) repeatedly solved our problems with inefficient protein expression.

Mainly due to the buffer component (phosphate buffer) the pH is kept stable which facilitates protein expression and folding. In addition, it is easy to handle - after inoculation you simply incubate the culture until harvest.

Did you include an affinity tag in your protein? Even if you can not see a band for your protein, you "could" still be able to purify 1-2 mg from 2 L of culture if you have an affinity tag like 6-His. I would not give up just because you can not see a clear band, and sometimes proteins run at the same position as E. coli proteins and so are not easy to see by SDS/PAGE gels when expressed at lower levels.

The suggestions by others on this post to either include glucose, or to use autoinduction media, may give you enough protein expression to see a band.

Michael, thanks for posting the Studier paper, the auto-induction technique looks really good, as does the addition of Mg to cultures for plasmid production.

U said that u have sequenced your gene and checked for frame also..Hope u have inserted your gene which is synthesized from cDNA.

Express your pet cloned into BL21DE3. and induce by low concentration of IPTG.

Check for ur specific protein in cell lysed supernatant and pellet also.becoz some proteins are insoluble, which wil present in pellet.

Hi Cahya, can you tell if the expression work with the OD at 2.0? Thank you!!! I have problems with my expression.