Hi there,

So your expressed protein is always insoluble. I would try different things:

1) lowering IPTG concentration

2) auto induction media

3) Coexpression of chaperones

Points 1 and 2 tend to slow down expression rate. Point 3 tends to improve protein folding efficiency.

Thank you Dominique Liger 

Definitely try auto-induction but perhaps also try Rosetta or Codon Plus cells to improve translation if it is a eukaryotic gene you are trying to express.

Switch the fusion to e.g. Trx, SUMO, Z-tag to improve solubility as the His-tag will generally not improve the solubility of proteins.

You could also try deteregents like Tween 20 or Triton (try 1%)  in the lysis buffer as some times this can pull apparently insoluble proteins into the soluble fraction. Is your lysis efficient or does your pellet still contain un-lysed cells?

Some recombinant proteins overexpressed in bacterial systems are simply insoluble. I would suggest keeping the pellet, gently lysing the cells under native conditions and harvest the protein aggregates. These can be solubilised in a number of ways, typically under denaturing conditions. Depending on what you do with the protein after, you will need to decide which conditions are going to be most benficial to your usage.

In our experience, altering IPTG concentration and time of induction do not change solubulity of overexpressed proteins in BL21 cells. 1mM seems to be best, and is suggested by the manual

If you protein of interest is of mammalian origin, I ll suggest you to go for mammalian expression host such as 293 or CHO cells. You will end up wasting lots of time and resources trying to express it as soluble protein in E. coli.

Add MBP tag to the list of fusion proteins. SUMO tag as mentioned earlier, has been also shown to be better than MBP and GST tags.

Since you are getting protein in pellet, it is definite that protein is getting expressed. If you want to stick with E. coli, you will have to play with slower induction as suggested earlier and may be get away from T7 promoter which is in pET30.

Firstly check that your protein really in insoluble, as suggested,  wash your pellet vigorously to make sure...you might be lucky. 

As also suggested you could use codon optimised strains of E. coli or strains capable of tolerating disulphide bonds

Lower temperature is also a good idea which you are already using

A different, weaker promoter may also work

In the past we have used different strains and different media: 2xYT, LB, Terrific broth etc

Make sure you are not leaving your cells to induce for too long. Take samples at varying time points to make sure. We have seen soluble expression early in induction then insoluble later on and eventually degraded product.

bw

Ian

I agree with Jeremy that changing IPTG concentrations rarely changes protein expression levels or solubility (at least for us). This is probably due to the lacY permease limiting IPTG entry and the T7 polymerase expression in DE3 strains being under the lacUV5 promoter which is essentially on/off. The only strains in which changing IPTG concentrations should really have an effect would be something like the Tuner(DE3) strains.

The addition of glucose (1% final) to all your plates and media may also help by blocking 'leaky' pre-induction during the early growth phase of your cells that could possibly lead to higher levels of mis-folding. N.B. glucose in the final expression media  is not compatible with auto-induction protocols but can be used in plates and starter cultures if your dilutions are >1:50.

As suggested using Codon Plus strains is of advantage if there are any rare codons in ur sequence. You can check by putting your sequence in Rare codon calculator which is available online.  If there are significant number of  rare codons you will get a truncated protein if you express in normal BL-21 . Hence you may use BL-21 Codon Plus RIPL strain which worked well when i tried to express my viral coat protein . E.coli grows well at 37C. So, try to check the expression by altering the levels of IPTG concentration 0.25, 0.5,0.75, 1.0 mM concentrations of IPTG. Check at which concentration you are getting optimal levels of protein expression. When we are using pET expression vectors, we get in precipitate only . I used pET29A and when i sonicated the bacterial pellet resuspended in PBS it was in pellet form or insoluble form only. I used to load it in SDS PAGE bigger gels ,destaining with 5% Ammonium bicarbonate and Methanol and gel extracted the protein by crushing in 5 %SDS   . Then, I dialysed against  10% sucrose  and concentrated.  Why you want your protein to be in   a soluble form only ?. What problem you have if you get it in insoluble form?