Out of these three, which strain should be preferred for expression of protein that has a tendency to form inclusion bodies?
If you have confirmed that it is in inclusion bodies than the expression works and thus you should not need rare-codons or disulphide bridges. Instead rather try lower temperature, lower concentration of IPTG, shorter time. Try to add sorbitol and/or betaine to the medium. That helped me when I was expressing my protein.
Hi Deekshi. I agree with Zdeno and Tomas. I induced the overexpression with 1mM IPTG at OD600=0,6 and then cultivated the bacteria overnight at 37°C at the begining of my experiments (I worked with BL21-CodonPlus-RIL strain). It resulted in the accumulation of my whole protein in inclusion bodies. I obtained the most of my protein in the supernatant only when I introduced some modifications: the induction with 0,1-0,5mM IPTG at OD600 up to 0,45, the RT and 5-7 hours as the cultivation temperature and time after the induction, respectivelly. The thioredoxin did not increase the solubility of another protein I worked with. Good luck :)
Hi there,
If you get inclusion bodies, you are in trouble with the speed at which the protein is produced. The synthesis is faster than the folding which results in agregation. In the lab, we systematically try:
1) Different strains (so I suggest you try the three strains you have)
2) lowering temperature during IPTG induction (15°C overnight instead of 37°C 3 to 4h)
3) Co expression of a set of bacterial chaperones at 37°C and 15°C (GroEL, DnaK...). You need to have the plasmid pKJE7...
4) Using auto induction media which allows a less brutal off/on transition than IPTG ...
Hope these will help in som ways...
i was used all of them, but my protein still could not express in soluble form, but BL21 Codon plus RP (DE3), it worked well ...
For me these three strains are obsolete; if you really need to control the level of expression, use the Lemo21(DE3) strain which produces lysozyme Y , also an inhibitor of T7 RNA polymerase but without any effect on the bacterial cell wall and you will get a normal growth. Avoid the strain with pLysE,
good luck
Dr Colette Duez
Hi Deekshi, Check in the literature if some researcher have already expressed your protein (or one related) in the bacteria that you plan to assay... to read this work http://www.ncbi.nlm.nih.gov/pubmed/23250226,... luck
Thank you Colette for the little advertisement of Lemo21 :-). I was just going to promote it. If you really want to optimize the yield of your overexpressed protein by regulating T7RNAPol activity, use Lemo21. This strain gives you the chance of finetuning expression to your needs. In particular difficult to express proteins (difficult to fold, membrane proteins, secreted proteins, etc) work better with this strain. For further information have a look at the attachment. I can also provide a protocol if needed. The strain is available through NEB. The other two publications may also be very helpful even though they deal with membrane protein production.
Article Optimizing E. coli-Based Membrane Protein Production Using L...
Article MemStar: A one-shot Escherichia coli-based approach for high...
I have no problem with BL21 DE3. But you should also tried to optimize the fermentation temperature because low fermentation may reduce inclusion bodies
With BL21-AI the T7 polymerase is under Arabinose promoter control and you can vary the concentration of arabinose in the medium for control of the expression.
I always express in BL21 (DE3) and I have no problems.
For the expression of the protein that I'm studying I don't induce with IPTG. I used a high copy vector (like pRSET) and let the basal expression of T7 RNA polymerase do all the work.
The conditions are: 18 ºC, 72-96 hrs, 220 rpm.
I know it sounds weird, but it works for me.
It is always hard to predict - but if you want to prevent inclusion bodies I would try to have lower overall expression. You could do this by more tightly controlling the promoter with lysozyme from pLysS or pLysE, or you could just change your induction conditions (like use less IPTG, lower the temp). Just try a few things on a small scale and see which works best.
BL21 (DE3) is a basic strain, neverthelles enalbles to produce recombinant proteins in soluble forms in many cases. The success greatly depends on protein nature although the optimization of culture conditions is also very crucial. I agree with the others. Try lower IPTG concentration, lower the temperature and optimize cultivation times. You can also try inducing the overexpression at lower OD600. It works well when you did not have strains with tight control and you observe a preinduction leakage. The auto-induction or chaperone coexpression are other options to avoid inclusion bodies formation.
Inclusion bodys are not necessarily a problem. It can help to purify your protein. After disolving in 6 M GuaHCl you can remove the Gua step by step by dialysis against buffers with decreasing GuaHCl concentrations. This should finally result in a refolded protein. If your protein forms disulfide bridges you have to add a redox-pair like cysteine/cystine 10 mM/1 mM.
I used BL21(DE3) with plysS induction at 20oC in M9 for soluble proteins that are prone to inclusion body formation- with lowering the temp I had no problem. Maybe you can even use LB or TB instead of M9
Right now I am working with T7express with pLysS from NEB, these are LacZ- and DE3 so I can look at function of my proteins - works but am currently having problems with protein toxicity that I will hopefully fix soonish! :)
Try out Lemo21 sold by NEB (see also our publication Wagner et al. 2010 in PNAS). This strain allows you to fine-tune the activity of T7RNApol and helps to achieve a strength of expression optimized for your protein of choice.
Article Tuning Escherichia coli for membrane protein overexpression
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Proteins