Thanks Walter, I will try this. Also, I get very low expression (200ug from 200ml of culture) but this is enough for my functional assays which work at the ng level. Now I read that storing at low concentrations also cause protein inactivation due to leeching of protein on the tube. I was thinking of adding BSA to increase the concentration and prevent binding to the tube. I will add a BSA only control in the assay but what are your thoughts on this method?

Thanks

Dear Anil, I have been looking at the sequence of the IL-4. Just to check: It contains a signal sequence (the first 25 Aa). For your cloning: I saw that you decribed the fusion protein as GST-IL-4. So, you removed the signal sequence (the first 25 aa, and then you took the sequence and pasted it to the GST, so that the GST is N-terminal? If you did so, the IL-4 will not be glycosylated on the Aa 65 as it should be (http://www.uniprot.org/uniprot/P05112) . There is no way for the cells to target it to the ER then to Golgi for glycosylation. It will stay cytoplasmic. The other thing: I am not sure that the Insect cells will be able to glycosylate properly trhe IL-4, even if you attach the GST to the C-terminal region for purification. This could be the reason why your fusion protein is not active. You could try to produce the IL-4 wthout tag, and by collect it by affinity chromatography. I hope it helps. Good luck

Hi Luisa,

Thanks for the comments. I actually used the entire IL4 cDNA sequence (all 4 exons including the signal sequence). The GST is on the N terminal end separated by a TEV protease site (for tag cleavage). it still contains the signal sequence so it should be processed properly (I think!).

I was also wondering about insect cell glycosylation as well. But I know that E coli expressed human rIL4 is not glycosylated but still has activity in these assays. Similarly, the guys who did this before used yeast cells and their functional assays worked.

Unfortunately, I need the tag because I am also expressing different variants of the protein so I cant use IL4 antibodies cause I dont know if they will distinguish between the different versions of the protein.

I am actully re running the CD23 assay at much higher concentrations but Im not too optimistic because the protein should be functional at much lower concentrations

Thanks again

I think you need the signal sequence before the GST coding sequence. The "original" signal sequence is i the beginning, what it makes me think that is not so strong to be recognized during assembly by the sec system in the ER if it is in the middle of the protein...

If the have used the E.coli or yeast, then you have one problem less with the glycosylation to worry about =).

In my experience, a myc tag works much better (it is really small, so no danger of "covering up" your active domain) and it has been my rescue in cases where I need to make a "ss+myc+cDNA(-ss)" combination for expression in eukaryotic cells.

The othe rproblem of GST, is that they tend to make dimers in non-reducing conditions. I say as well that your IL-4 has a couple of sulfide bridges... it could be a wrong folding at the moment that you try to purify it... many variables, I know!

until later...

Luisa