How did you cheked that your protein was not overexpressed? Maybe you should use E. coli DE3 pRIL strain with rare codons plasmid. Maybe expression lvl is so small that you can not see your protein on standart sds-page gel. Or you tried Western blotting?

I lysed my cells using BugBuster and performed SDS page for both the supernatant and cell pellet at all the hours tested. Even then, no expression was observed.

Hi Dawid,

Nop. Have not performed Western Blotting.

A few questions/suggestions about your expression:

1) How have you confirmed that you have no expression? Do you have an antibody against the protein of interest that you can detect it with? How are you trying to purify the protein?

2) As Jiyong says, have you checked the insoluble fraction for expression also?

3) I would certainly say that it is worth trying to add a tag - why not use the C-terminal His-tag available in pET-21 as well as trying the N-terminal His-tag in pET-14?

4) Given that you have some rare codons present, you could address this issue, either by using a strain providing rare codon tRNAs (e.g. Rosetta2, BL21 codonplus etc.) or by getting the gene synthesised with the codons replaced with more common ones.

5) You could also try other tags, of which there are many. One which I have had success with is the SUMO tag, available in many vectors (e.g. those from LifeSensors). Other tags such as GST, MBP, ... could be tried.

6) You may well find that in spite of these approaches your protein will not express successfully in a prokaryotic system and you may need to think about a eukaryotic system.

For any protein expression, you really need to do a Western blot at this early stage. Unless the protein is very highly expressed you will not necessarily see a band just in the cell lysates. Addition of a tag provides a facile way to blot for the protein if you don't have an antibody to the protein-of-interest available. Western blots will also help you follow the protein throughout the expression.

Having looked at your temperatures, I would also try expression at a lower temperature of e.g. 18-22°C overnight, following growth at 37°C to OD600=0.6-0.8 prior to induction.

So there is no expression even in the form of unsoluble proteins (inclusion bodies)?

I think the problem is very likely because of your rare codons (if there's no expression in soluble nor insoluble fraction). In that case, you should try the strain with rare codons plasmid, like Dawid suggested.

Also, it is true that addition of fusion tag can improve the expression of proteins, but mostly if the problem is that your protein of interest isn't soluble. In that case, expression at lower temperatures (16 C) over night with lower IPTG concentrations (o.2 or 0.5 mM) also helps.

For Q1: its hard to say if a protein will or will not express with N-terminal his tag unless you try. I would carryout additional tags like trx or GST or MBP in parallel to His tag during cloning to save time.

During subsequent expression I would take in to consideration of the strains suggested above to overcome rare codon problem.

I also think its good to use these strains first and check the expression your protein again. If this doesn't work then you can go to cloning.

first step: did you sequence the vector with the insert? (colony-check-pcr for me is not sufficient), if you are sure that the sequence will be right, than you can do expression.

next step: how will you purify your protein without a tag? did you use an antibody-column? than do an western-blot with this antibody.

i agree completly with roger, you need a western-blot, i prefer to use a His-Tag and use a His-Tag antibody to check the steps of expression and purification.

For a lot of proteins we used the pSumo3-Tag too and it worked very good. I prefer this tag together with Rosetta gami 2 E.coli cells for expression.

Yes I forgot Sumo, its a good one. I have good success with it. Thanks Ties.

Also everyone who have answered including me have assumed that sequences/ORF are confirmed by sequencing before moving to expression.

Hi Saranpal,

Lots of good suggestions, but first have you determined what reading frame your gene is in and then looked to see if it is, or is not, in-frame with the vector's C-terminal His-tag?

The issue with rare codons and low protein expression levels comes into play "usually" when you have multiple rare codons in a row. Since you do not have that I would not focus on rare codons right now.

Do you have any reason to believe your protein may be toxic?

The His tag is primarily to aid purification of the construct and in most cases will not otherwise aid in expressing a protein that does not or is difficult to express. There are other fusion tags available that help express folded/soluble proteins. They might be helpful if you see some expression in the first place (say in soluble fraction or in inclusion bodies). The other thing to try would be to have a codon optimized genetic sequence to start with in combination with different fusion tags. There are many good publications which report findings with different tags. Thioredoxin, SUMO, MBP, NUSA are few popular choices.

Hi, did you check that your construct is in frame?Your protein may be toxic , you can add glucose to the media( for plates too) , start culture for expression from the fresh transformed colony and try to express at low temperature( 16C) and low IPTG( 01mM) concentration. Rosetta competent cells may help too. Good luck Tanya

Hi all,

Thank you very much for your esteemed and critical comments pertaining my protein expression problem. I have taken your advise on designing primers for pMAL vector, which has a MBP fusion. Additionally, I will also be performing my expression using the pSUMO vector and on Rosetta soon.

As for the upcoming week, I will be doing expression at low temperature (18C) and low IPTG concentration just as pointed out by Roger and Irena.

To the questioned imposed by Totyana and Yogesha on whether my protein is in frame or not?

Yes, I have sequenced my vector with insert, and it shows that it is in frame.

Once I have successfully expressed the protein, I will definitely alert you all here.

Once again thank you very much for helping me troubleshooting. :-)

Well Saranpal I too have a similar problem right now and am planning to add glucose to my medium and also freshly transform BL21DE3 cells and look out for expression