Human coronavirus VLPs is composed out of 3 subunits with transmembrane domains, most likely co-purifies with membranes. The particle co-expressed in baculovirus is not soluble when purified in 50mM TRIS pH=8, 100mM NaCl. Please give us any tricks, advice, or negative experience will led to high scale purification set-up.
I use for inclusion bodies alkaline solubilization, maybe you can use this method. Regards
Article Design and Optimization of a Purification Process for MY32/L...
Dear Carlos, I need the native conditions to keep the multimer stable...
Dobry wieczór, Jan! Proszę, przeczytaj ten link. może to pomoże. Miłego weekendu,
hhttp://cdn.intechopen.com/pdfs-wm/32429.pdf
ttp://fivephoton.com/index.php?route=product/product&product_id=78
I was once overproducing viral protein, they also formed inclusion bodies and I was able to solubilize tchem using two tricks:
1. I have decreased the rate of protein expression - however, I have produced my proteins in bacteria and it was easy to do, I have just switched from IPTG to lactose, it decreased how fast the protein was produced by ab. 5 times and the inclusion bodies which still formed were much more loose (before, I have needed 6M of urea to solubilize them and these are not a native conditions for sure); check if You can do similar thing with your baculoviral system
2. I have done solubilization of such more loose inclusion bodies using sodium lauroyl sarcosinate (INCI), also known as sarkosyl, an ampiphilic ionic surfactant derived from sarcosine; I have used 0.5% of sarkosyl solution in normal buffer for proteins, shaked it for 3 hours at 22 centigrades and was able to observe expected trimers of produced protein during filtration on Sephadex column.
Method is very old, I have done it about 20 years ago, but maybe it will be helpful as not a lot of people think about making the expression system a little less efficient so the host cells do not need to scramble as crazy to try to chaperone overproduced proteins in the right shape. And sarkosyl is not often used as a solubilizer and it is much better for the next purification steps than SDS (or urea).
Dear Jan, you can try alos in situ methods like PLA assay for investigation intact complex.
I hope this will help!
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques