You do not list your expression conditions. In http://europepmc.org/articles/PMC3303126;jsessionid=jCnQBeoOb5GpLoZ6zn0e.0, the reference cited for  UniProtKB - Q5JVF3 (PCID2_HUMAN), the proteins were expressed at 20°, I would try to follow their conditions as closely as possible.

Thanks, Annemarie, i used 0.2 mM IPTG, and induced at 18 degree overnight. I also read the paper you mentioned,  they determined the crystal structure of PCID2 (residues 205-399) complexed with DSS1 (residues 38-67), and this paper said coexpression with DSS1 was necessary to obtain soluble PCID2.

So, next I may start co-expression, insert two plasmid into one vector.

I agree with your plan to co-express with the partner protein, from the same plasmid - that is likely to help a lot. 

The structural paper only expressed the second half of the PCID2, so it's probably best to start with that.

I would also suggest reducing the amount of IPTG that you are using. Adding to 0.2 mM is pushing the T7 expression system to not far from its maximal amount. You could easily titrate this down to 20 uM or even 2 uM. Sometimes less is more - reducing the amount of protein produced can result in some of it folding. I have had good examples where this has turned an insoluble protein into one that gives several milligrams per litre of soluble protein. A test at small scale can save a lot of pain further down the line.

Good luck!

Hi, I can see you are expressing human protein in bacterial system, probably that is the reason may be not folding properly and forming inclusion bodies. Did you try autoinduction? if you haven't done, I would do try first. Second try to express in yeast or Pichia system. Good luck

Thanks for all your answers, my lab only have E.coli. system.

I will follow Nicholas's suggestion to titrate the IPTG amount from 10 uM, 30 uM, 90 uM in small scale.

And autoinduction from Thamarai maybe a good idea, I never heard about it, and I checked the autoinduction protocol,  we do not have some required medias,  this maybe not my prior choice. If i fail with coexpression, i maybe try autoinduction.

Autoinduction is a very powerful technique - we generally use it as the "first pass" approach, as when it works, it works brilliantly, and delivers more protein for less work. That's always good. Some proteins seem to express much better in AI media (it's probably as the glycerol in the media changes the osmotic pressure on the cell), compared to traditional media.

However, the AI approach is pretty much equivalent to using 1 mM IPTG or more - so the T7 system is working at maximal rate. This can be a significant issue with tricky proteins. It's almost always worth trying, but is not a cure-all.

You don't need to buy the auto-induction media - the recipes for running it are all detailed in the paper linked to below. I've extracted the relevant protocols and used them for a decade with some great results.

http://www.ncbi.nlm.nih.gov/pubmed/15915565

Dear Zixun,

some times, no matter what you do, some proteins just end up in inclusion bodies for reasons that we don't know. In that case, the option(s) you are left with  is(are) to isolate your protein from inclusion bodies and to refold. Some times the proteins in inclusion bodies are well folded as well and its well documented too. We had similar experience and got success with methods that utilize mild detergent conditions to solubilize the protein from inclusion bodies. You can try our protocol we published recently to isolate plant Cellulose synthase from inclusion bodies. 

see the attached paper for details of the protocol.

Thanks and good luck

Venu

Dear Nicholas and Venu, thanks for your information very much, i will try auto-induction and refold as you suggested in the future, if i succeed, i will reply here, thanks again

thanks very much, Kristian, i will discuss about the yeast expression with my supervisor in next group meeting.

Dear all, i decide to try yeast expression system by using pichia

Hello Zixun,

                      If your protein of interest does not have any post translational modification to do, then i think you can still express your protein using BL21 system, According to my experience, when most of the protein goes into inclusion bodies i have following points to look into.

You can use above 3 conditions in various combinations, as they will reduce the rate at which your protein is expression dramatically, giving ample time for its folding.

If still your protein is in inclusion bodies, then you have to go for protein refolding.

But for protein refolding you have to consider following points.

Hope this will help you in getting atleast some portion of your protein in a soluble form.

dear Manojkumar, thanks for your answers so much, actually, i already did lots of works about refinement of expression conditions, including low IPTG around 2uM, 10uM, and low temperature at at 16 degree, but did not try 8 degree, i worry the cells cannot grow in 8 degree. and i also try to add IPTG at OD1.5, but still they are inclusion bodies.

now my main idea is to use yeast expression system, at the same time i will also try to refold from inclusion body, because i found many papers used the protein purified from inclusion bodies and did crystallization and solved structure.