Hi Anil,

There're two major possibilities.

1. Lysis buffer: 50 mM KCl is on the low side. Use at least 150 mM salt (NaCl or KCl). Some proteins form polymers at low salt concentration (50 mM is low). Is your protein known to bind Ca2+ and Mg2+, if not, try skipping them. Triton and BSA are also not necessary agents in the lysis buffer. Detergents such as Triton aren't too useful in "solubilizing" misfolded (as Olga suggested) and supposedly soluble proteins.

2. Domain boundary of your constructs. Do they code for full length protein, or part of it? If former, you're probably missing the obligate binding partner(s). If latter, you may want to re-define the boundary.

HTH

waiching.

Hi Anil,

I would also recommend doing a time course expression post-infection (eg t24, t48 & t72 hrs using a range of MOIs) and choosing the condition that provides most soluble protein. In many cases the overall yield is less but the ratio of soluble to mis folded /insoluble protein increases. Another possibility is co-expression with a chaperone to try and raise the level of soluble protein. I often see cell type differences too so worthwhile trying Hi5 cells for the expression trials.

Good luck!

Eddie

Thanks everyone for the helpful answers. One additional thing that may be going on is that the folding of the mutant protein may be exposing hydrophobic regions or associating with membrane as well causing the insolubility. Or it may very well just be misfolding. In the lysis buffer, I am going to try to increase the salt concentration and perhaps try a zwitterionic detergent like CHAPS which apparently does increase solubility (I found in the literature). I have tried different MOIs and timepoints and found the optimal at MOI 10 and harvesting 2.5 days pi. One thing is that the cells were almost 100% viable at the time of harvesting. I have two further questions:

1. In your experience, is there a relationship between viability and protein expression/solubility i.e. at what viability is the best to harvest cells or is it different for different proteins?

2. Does anyone have any experience with adding (to the lysis buffer) different additives to increase solubility and/or expression? the literature suggests things like Trehalose (an osmolyte) of arginine (chemical chaperones) but these are geared more towards bacterial expression systems

Time is of the essence and I unfortunately don't have time to make new constructs containing molecular chaperones but I may also try a different cell line as well

Thanks again

Anil

Hi Anil,

Can I ask the intended end use of the protein? Do you want just a lot of soluble protein (which can be "soluble aggregate" and non-functional), or something for biochemical analysis?

waiching.

Hey Wai-Ching,

I actually need the protein functional as it will be used in immunological assays (protein is a cytokine isoform). The problem is while I am getting soluble protein, I need a bit more to do some of my assays so anyway to increase the yield would be helpful. I am actually busy now testing the function to ensure the soluble form is working!!

Hi Anil,

Cytokine, that should be a secreted form? They normally are. You would be collecting your protein from the culture media, and there should be no need to worry about cell lysis. I presume you have a secretion signal at the N-terminal end, and some sort of purification tag at the C-terminal end?

waiching.

so the wildtype protein is secreted into the media. However the isoform (which is missing an exon) is not secreted into the media but remains in the pellet. The isoform probably folds in such a way so as it is not secreted. As such I had to redesign the DNA sequence to remove the signal sequence in the isoform. It does carry a 10xHis tag on the C terminal. I also tried a GST tag but in insect cells there is co purification of an intrinsic insect cell protein and it was very difficult to remove.