We have been trying to purify a chimeric of MW, 31kDa in insect and mammalian cells using the pOET5 and pTT5 vectors respectively. Unfortunately, we can detect very low levels of the protein in the supernatants through Western blot, and any attempts to purify using SEC reveals that the protein is forming aggregates. Can anyone help with a protocol to handle this kind of situation.
Hi Robert,
The MW of your target protein is not very large, and the mammalian expression system is used, so it is likely that the properties of the protein cause aggregation, provided that the purification process is performed correctly.
1. Is the target protein soluble or membrane protein? In the case of membrane proteins, an appropriate detergent needs to be added.
2. Is the target protein full-length or truncated? Is the truncated position appropriate?
3. Did you try to use solubility-enhancing tags?
4. Is there any protein that interacts with the target protein under endogenous conditions? It is recommended that you consult the literature, in my experience, some proteins need to be co-expressed with interacting proteins in order to stabilize their conformation.
Yours truly
Dear Robert Adamu Shey
i thin that to help you we have to know more details about the proteins and the expression sistem and purifciation conditions that you are using.
Just a preliminary question:
what do you mean for a "chimeric"?
Is it not a single protein but a fusion of 2 proteins?
if yes. Are you able to produce with the same expression sistem the single protein ot aggregates?
best regards
Manuele
Hello Manuele Martinelli and Roger Davis , thanks for the answers. Just to provide more information on the proteins. The protein is multi-epitope chimera (designed by combining several epitopes from different proteins). Our analyses reveal that no TM domain is present. We did not express the protein with any solubility tags and we also found out that the protein interacts with some host proteins.
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Pharmaceuticals
- Drugs