Dear April

I think that some of the proteins are difficult to express in soluble fraction of the lysate, Better you can try low post-induction temperature (15-20 degree) with short time (30 min - 2 hours) and high IPTG concentration (5-10 mM) . If you get some of your protein in soluble faction, that's will be nice one. Other wise you have to follow the denaturation-cum-folding your protein.

Hi Naveen,

Thank you for your response. I have tried several induction conditions including a series of IPTG concentrations as well as induction times and temperatures. I am specifically interested to know if anyone has tried this particular strain of BL21, soluBL21, for protein expression and if the strain allowed them to purify previously insoluble proteins.

Thank you.

@Naveen.. I want to know that at 5-10mM IPTG conc will not affect cell survival as I read somewhere that more IPTG conc is toxic to bacterial strain.

I have tried an IPTG concentration series from 0.1mM to 1mM and found that it is already maximal at 0.1mM, with some expression already coming from uninduced cells. I was told that this is common and even lower induction concentration or no induction at all may help protein solubility. However, this did not help in my case. Thanks for the comments.

In my hands this problem is either solved by (A) a fusion tag, such as MBP, or (B) lowering the expression temperature; If that' doesn't help, it becomes really unlikely that anything else will keep it from misfolding in E. coli. You may see minor effects with strange commercial strains, codons, chaperones and what not, like improving the soluble fraction from 1 ng to 1.5 ng. But it's not usually a magnitude that you care about in a practical sense.

If fusion tag and lower temperature don't do it, in most cases the you'd have to ditch e. coli altogether for this protein.

Hi April,

I am excited to see your question about SoluBL21 cells, as today I am actually using these cells for the first time myself. I will let you know later in the week how things turned out.

In contrast to some of the other posters, my own experience has been that the specific cell line absolutely has the largest impact on whether the protein expresses solubly or not. In my own work, the cell line has always had the single largest impact on soluble expression, much more so than fusion tags or expression temperature. I am currently working with several proteins that express very well in E. coli BL21, BL21 Star, and Rosetta 2 cells, but unfortunately 100% in the insoluble fraction. Whenever this happens, my next step is always to turn to E. coli C41 or C43 cells (sold by Lucigen). For my current proteins, expression in both C41 and C43 cells leads to 100% of it being in the *soluble* fraction, although the overall yield is noticeably reduced compared to BL21 cells. However, I'll take soluble protein in lower yield over insoluble protein at high yield any day of the week! In fact, the reason I'm trying SoluBL21 at all is to see if I get a higher expression level compared to C41 and C43 cells. I'll know later in the week if the protein is soluble upon expression in SoluBL21 and how the yield compares to C41 and C43 cells. Good luck with your own protein expression!

Wow, thanks Matthew! I look forward to hearing your experience with the cells. I should get my hands on C41 and C43 strains too, thanks for mentioning them!

So, I gave the SoluBL21 cells a try, and I must say I was disappointed. I could never actually get them to grow! Despite using their recommended formulation for M9 medium (which I use all the time anyway) and despite following their growth protocol, I could never get the cells to grow in liquid culture. I was able to transform and plate them with no trouble, but they never grew in culture. And yes, I'm sure I was using the right antibiotics and made the media correctly, etc. Very strange. I'll give it another try again eventually, but I have to put it aside for right now. I'll stick with my C41 and C43 cells at present.

Strange! Thanks for replying. I guess I will hold off of purchasing them for the time being... Good luck, and please let me know in the future if you figure out the problem.

Hi April,

So of course every protein is different and what works for Matthew may or may not work for you.

I was able to get the SoluBL21s to grow fine. I did starters at 37 in LB and 400mL cultures at RT in MagicMedia (Invitrogen) from 1:100 inoculation. Kan selection. I'll post back when I know the end results. i.e. if the SoluBL21s actually do a better job than normal BL21s.

Hi Everyone,

I am also struggling with same problem that April has mentioned.  Despite changing a lot of things like conditions and media composition I have not been able to get the soluble protein.  Please let me know your experience with SoluBL21 or any other E.coli strain that can resolve this problem.

Thanks in advance.

Hi everyone,

So recently we have purchased the BL21 Sol in our lab but we have serious problems in liquid media when we inoculate a colony the growning bacteria inside the liquid media are pieces. The strange is that these pieces continue and multiplied, but without any cloudy display as usuall bacteria. I would like to ask you if any of you had the same experience.

Thanks in advance.

I have used SoluBL21 with mixed success. With some proteins, it does increase solubility, with others it doesn't. It is a matter of trial and error. As to growing cells in liquid, I experienced no problem except that the cells tend to form clumps more readily; however I managed to grow recombinant cells in M9 medium for labelling with selenomethionine

Pierre Béguin could you maybe share your protocol?

i am trying it now as Genlantis describes it in the delivered protocol, and it is more a matter of luck if the one-colony overnight culture growths or not

I use no special protocol nor precaution with SoluBL21: I pick isolated colonies from plates and inculate them into liquid medium. I must confess that I got the strain second hand, so everybody else's troubles begin to make me doubt whether I got the genuine SoluBL21. All I can say is that my recombinant strains grow somewhat slowly, that transformation efficiency of CaCl2-prepared competent cells is poor, but sufficient for transforming whole plasmids, and that it forms clumps. Therefore, when I transform, after the no antibiotic expression step , I vortex the cells vigorously before plating. I may add that I did notice better soluble expression with some, but not all recombinant proteins that I tested, so I assumed that the strain was SoluBL21, but I have no proof.

Maybe the best thing would be to ask the technical staff of Genlantis directly.