We were working on purification of a his-tagged protein from Expi-CHO cell pellet and tried different buffers along with sonication to get the protein out in the supernatant for further purification with Ni-NTA columns. But the protein remains in the cell pellet.
I've tried using two different buffers for resuspension of the pellet:
I was wondering if anyone could have some idea about how to lyse the cells and get the protein in the supernatant.
Thanks in advance.
It frequently occurs that an overexpressed protein ends up in an insoluble form because it does not fold properly during expression.
There may not be any way to make such a protein into a soluble form except under denaturing conditions. The protein can be extracted from the pellet with a strong denaturant such as 8 M urea or 6 M guanidine-HCl. It can also be purified under such conditions. After purification, it has to be refolded. It can be difficult and time-consuming to find the right conditions for refolding. Therefore, it may be preferable first to explore other options for expression of the protein.
For example, you can try different heterologous expression systems and in vitro transcription/translation. You can try making fusion proteins, in which the protein of interest is fused to another protein that has high solubility (such as maltose binding protein, glutathione-S-transferase, or SUMO), or coexpression with chaperone proteins.
Dear Smriti Chand
is it your protein predicted to be a membrane protein?
Iif yes, the protein is not re-solublized since you need to add some detergents to extract it from the membranes.
The most used detergents those are reported to be non denaturing used to my knowledge for this purpose are DM, DDM or OG, which unfortunatelly are quite expensive reagents.
good luck
MAnuele
Thanks Manuele for the answer. The protein is potentially not a membrane protein.
DEar Smriti Chand
in the sequence is it present an N-termjnal signal peptide for protein sectetion in the surnatant?
best
manuele
Hi Manuele Martinelli,
The sequence does not have a signal peptide in it. But our previous results showed that the protein secretes in the supernatant (in the transfected HEK-293T cells).
Thanks
Smriti
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