I can suggest some factors considered: 1, the HLA and B2M both inclusion body expression, wash the proteins as pure as possible, probably above 90%, added with a Q-HP intensive washing. 2, dilution refolding, HLA: B2M: peptide 1:2:10 probably, firstly dissolve the peptide in the refolding buffer. Add the HLA and B2M simultaneously or firstly B2M then HLA. Refolding takes place in 4 ℃ and the refolding buffer should be pre-cooled at 4 ℃. Refolding o/n. 3, FPLC sorting. Probably can add a step Q-HP enrichment. The complex may elute at about 200-300mM NaCl, when this occur it often suggest good refolding. 4.SDS-PAGE non-reduced to test the ratio HLA:B2M. 4, Probably viral peptide antigen is easier than tumoral antigen. Wish you a success!

I think the answer is a very simple one, if I've read your protocol correctly (and assuming your peptide is ok).

You don't mention a reducing agent anywhere in the protocol. Your IBs are dissolved in "Denaturing buffer: 8 M urea, 100 mM Tris-HCl pH 8".

This will not break any disulphides that have formed or misformed between chains during expression or preparation. Furthermore, any free cysteines present will quickly have oxidised to their cystine forms. Since each of the disulphides that hold an HLA together are formed between the thiol groups of two cysteines it will be problematic if you don't have any!

I would suggest adding either DTT or TCEP to your chains when you've mixed them in their denaturing buffer (prior to refolding) and give them about 30 minutes to ensure thorough reduction. Then proceed with your refold, and see if that has helped. Personally I'd get some EDTA in there too, as you had Mg and Mn in your lysis protocol, which are loved by proteases.

Also, another thing that you might be doing (inadvertently) is using SDS-PAGE buffer with mercaptoethanol or DTT in it when you run your gels. That could make you think you have no product - but I doubt you've done this as actually you would not see aggregates either if you had.

In any case, make sure, when running gels, you run two lanes for your products, one with mercaptoethanol or DTT in the loading buffer, and one WITHOUT, so that you can confirm the correct components in your product.

Hope that's useful.

John Hinks Thanks for your response. I did not mention DTT, but we have been adding after each inclusion body wash step and prior to dilution. We are thinking the problem is our peptide.

Min Li Thanks for your suggestions. We are working on purifying our HLA alpha chain further!

Hi Omar, sounds like it could be the peptide then. I know that HLAs are very unstable without peptide bound. A possibility might be a peptide exchange whereby you refold in the presence of a well established but fairly low affinity peptide present in excess in your buffer, and then once folded you exchange into a solution with the desired peptide (assuming it has a higher affinity and displaces the original peptide). Good luck.