Hi Mary,

This is a common problem.

1) You can use different strains for efficient expression of (potentially) toxic / heterologous proteins; you tried some, but I think there are some even better suited to this aim.

2) Have you codon-optimized your ORF for E.coli?

3) Choice of the type and localization of the tag could make a HUGE difference! Instead of bulky GFP, problematic 6xHIS, you may opt for HALO or FLAG-tag.

4) Have you ensured you cloned FULL coding sequence?

Hope this helps! :)

Hi Mary,

This is a common problem.

1) You can use different strains for efficient expression of (potentially) toxic / heterologous proteins; you tried some, but I think there are some even better suited to this aim.

2) Have you codon-optimized your ORF for E.coli?

3) Choice of the type and localization of the tag could make a HUGE difference! Instead of bulky GFP, problematic 6xHIS, you may opt for HALO or FLAG-tag.

4) Have you ensured you cloned FULL coding sequence?

Hope this helps! :)

Hello Mary,

check for the following issues. 

1. if you have cloned the whole gene,from genomic DNA, it may not get expressed properly as there can be splicing issues. So, try making a cDNA and then clone it into a plasmid.

2. Also, as marcin said, try to check whether you've cloned the whole coding region or a fragment of it. If length doesn't match, try optimizing the PCR protocols.

3. Choosing site for cloning is also a factor. Fusion proteins are difficult to generate. Choose your sites wisely.

Hope this helps. Good luck. 

Hi, I agree with the people's opinion above and I wish to add that you can use E. coli KRX strain which is rhamnose inducible and is working Ok for me.

sometimes mutation in regulation sequence in vector can stop production. after trying different concentration of IPTG, temperatures, and  strains, maybe there is problem in vector 

Thanks everyone for your replies. The full gene was cloned from cDNA. Marcin, could you advise me some more bacterial strains for expression? Also, how can I determine if my protein is toxic? My cell growth looks normal and the cell density is good after induction..Anubhav, I did not understand the choosing of sites point. Could you please elaborate it a little? Thanks a lot again.

In case of toxicity try the TBL21(DE3)pLysS and BL21(DE3)pLysE strains, but as far as I know there is no way to know in advance whether the protein of interest is toxic other than by literature.

Please see: https://www.google.com/webhp?sourceid=chrome-instant&ion=1&espv=2&ie=UTF-8#q=E.coli%20strains%20designed%20for%20expression%20of%20toxic%20proteins or the attached file.

BL21 or Yordan's KRX are good examples, although your DE3 seems to also be belonging to this group 

You also might want to consider alternative cloning/expression systems (restriction vs. USER or TA/Gateway; the latter of which helps ensure the in-frame cloning of your ORF). It seems you might have a problem there with proper reading frame rather than with expression itself; for such reasons, I do not use restriction-based cloning, only if no alternatives exist.

GOOD LUCK! :)

Hello Mary,

regarding choosing the sites of cloning, sometimes, due to frame shifts, orf is not fully complete and the sequence may have an unexpected stop codon in between. Just check for that. Also, sometimes, cloning distorts the promoter sequence, which may also hamper the expression. Try to clone your sequence,  100 or more base pairs downstream of your promoter.

Hi there,

Have you got any idea of the biological activity of your protein?

If you don't get anything from pET system, try other systems maybe less efficient in terms of production yield but which may possibly allow you to get your protein expressed (lac, ara promoters)...

May be auto inducible media from Studier can be the answer (lactose instead of IPTG as an inducer)...

If it is possible, you may try to use  TnT® Quick Coupled Transcription/Translation System from Promega to quickly make sure you indeed had a workable plasmid construct if you see the protein on western blot.  IF you failed to see any protein product, then you had to use a different expression plasmid at least.

Some host strains, e.g. C41 (DE3) and C43 (DE3), deal better with membrane proteins than it parent BL21 (DE3). If you know the sequence of you protein and see that you are having problems, another way to get the protein is by synthetically synthesized it as a peptide. Here is a guide for you to take into consideration: https://www.neb.com/tools-and-resources/feature-articles/bypassing-common-obstacles-in-protein-expression

Good luck.

There are numerous additional techniques that you can try out to increase soluble protein expression, here are a few suggestions:

- Examine both cytoplasmic and periplasmic expression using various signal sequences as the ideal redox potential varies for each protein.

- Test additional promoter systems such as PhoA.

- Test different vector backbones who's origin of replication generate either high or low cellular copy numbers.

- Co-express with chaperones such as GroES and GroEL.

- If there are numerous cysteine residues, co-express with a disulfide bond isomerases such as DsbC.

Unfortunately no one technique works for all proteins, so it is a matter of trial-and-error. Hopefully one or a combination of a couple of these suggested techniques works out for you.

One more issue remains to be addressed: Please let us know, whether it's a soluble or membrane protein (prediction).

Otherwise, the only one that you could address would be the codon optimization (mammalianE.coli).

Thanks for all replies! Will try out the above suggestions. My protein is not a membrane protein; but its a fibrillar protein..Could that be a reason for non-expression??

Hi,

Please check for the frame properly again and also for stop codon. I feel problem lies in cloning.

if everything else is fine in cloning you can try using codon plus host or else use codon optimization method. have you checked at lower temperature???

check your ORF and confirm by western blotting some time protein may poorly express or not resolve properly on SDS-PAGE

Also,

Based on all the info in this thread, the likely suspects are in-frame cloning and/or codon usage. If it keeps not working @E.coli, you could always transform yeast or Agro-transform some plants. Good luck!

Check whether the orientation of gene is correct  .Í suggest you try to express the protein in mammalian system sine it is mammalian protein?

all the best