E.coli BL21 DE3 is not RecA- stain which means that if your protein is 'toxic' to the cells they may prevent its expression through recombination (i.e. your protein is unstable). To overcome this you might try using a strain that has a pLysS plasmid to lower the background expression level of target genes. At the same time if your protein is mammalian in origin you might benefit from a strain that also expresses rare tRNAs (codon usage is different in E.coli and mammals). I also generally prefer to always start an over-expression by freshly transforming my expression strain with a plasmid (sequenced) and rather then go to a plate go directly to 10 ml liquid culture which I then use as a starter for a 1 L over expression. ALso I have good experiences with using low temperatures (i.e 15 ˚C -after IPTG addition), low IPTG conc. and long expression times. There are also autoinducing media that work very good and have low conc. of glucose to initially repress background expression.

Hope this helps

If you check that your gene is expressed (as mRNA by using eg. qPCR) then the likely explanation is that it is cleaved post translationally. If that is the case then you may want to try another tag. Can you express your protein w/o a tag? That information would also be important for you to know.

While prokaryotes do not (seem to?) contain the complex membraneous organelles similar to eucaryotes that by no means equals that there would not be PTMs in bacteria.

I think you gene of interest is not in the same frame as that of GST. I think you are using N-terminal GST tag. you can try C-terminal tagging. I hope you will be able to express them if you put your protein in frame.

thank u all...

sequencing results show that there is no stop codon or frame shift...

yes i am using N-terminal GST tag and since i wish to use affinity chromatography for purification, i have to use a tag... my protein does not get expressed with his tag...

E.coli BL21 DE3 is not RecA- stain which means that if your protein is 'toxic' to the cells they may prevent its expression through recombination (i.e. your protein is unstable). To overcome this you might try using a strain that has a pLysS plasmid to lower the background expression level of target genes. At the same time if your protein is mammalian in origin you might benefit from a strain that also expresses rare tRNAs (codon usage is different in E.coli and mammals). I also generally prefer to always start an over-expression by freshly transforming my expression strain with a plasmid (sequenced) and rather then go to a plate go directly to 10 ml liquid culture which I then use as a starter for a 1 L over expression. ALso I have good experiences with using low temperatures (i.e 15 ˚C -after IPTG addition), low IPTG conc. and long expression times. There are also autoinducing media that work very good and have low conc. of glucose to initially repress background expression.

Hope this helps

@sean : thank u... i will try with plysS and rosetta gami (for rare codons)...

If its of mammalian origin you may have to check the codon usage. There are a number of codons that are used in mammals that don't translate well in E-Coli. Other than that it can be degradation or as CN said your protein is not in frame.

Hi Archita,

I hope that you had did the codon optimization before you synthesis the gene. If it is cDNA from mammalian origin then there is high chance of rare codons present in the gene of your interest. You can detect it by this link http://www.genscript.com/cgi-bin/tools/rare_codon_analysis

hope this helps you

I would first double check that the bacteria have your plasmid and not just pGEX-4t2, since that would explain your results. You can do a mini-prep of the BL21 cells to isolate the plasmid and then do a RE digestion to check for your insert.

Have you sequenced across the GST-thrombin-your gene junction to make sure there is a continuous open reading frame?

You mentioned that your protein "does not get expressed with His-tag", that could point to your gene expressing a toxic protein. E. coli can do all kinds of things to plasmids that encode toxic genes. Checking that the cell lines still contain the plasmid "with your gene" wold be helpful.

I agree with the comments given above. You need to double check your plasmid for the presence of your target gene.