From transient e get very high levels of secreted recombinant protein with a N-terminal his-tag, but our purification results are poor (less than 5% yield). We have tried desalting the supernatant, using 5ml or 1ml HisTrap FF or HisTrap excel columns, with or without Imidazole in the loading buffer, but so far there is hardly any effect.
Expression and purification is determined by another tag (c-terminal) on the protein which we quantify on a bead-based assay.
Are other proteins being retained? What immobilized ions are you using? Buffers? EDTA? If could be that the tag is somewhat hidden. To disregard this, add urea to denature the proteins and run the adsorption-desorption.
One possibility is that the N-terminal His tag has been lost due to proteolysis.
Are you using serum-free medium during expression/secretion, or medium containing fetal calf serum? The presence of a high albumin concentration from serum could be hindering binding of the protein to the Ni column.
Thank you all for the answers.
It seems that some other proteins might be retained.
We use Nickel ions. interference with low MW substances have been excluded as desalting/dialysis has not really improved the purifications.
We are using serum-free media, so proteolysis is possible.
We would actually rather not move the tag to the other terminus, because this way we can be sure that the protein is complete if we detect both tags on N- and C-terminus.
We have not tried using urea for this, will maybe try that soon.
Hello Christiane,
I have the same problem than you with purification from expi293 cells. Do you find a solution ?
Thanks
Hi Emilie,
I have not really solved the problem, but found a different way to separate my proteins of interest, based on size (using a Sephacryl S-200 column). My co-worker has got some improvement for other affinity-based purifications from ExpiCHO supernatants after extensive dialysis prior to purification.
I hope this helps a bit.
Christiane
Hello Christiane,
Ok, I try once to dialysis my medium against sodium phosphatebuffer in a vivaspin column but it doesn't work. Can you ask to our colleagues what system of dialysis and buffer they used ?
Thanks
We use dialysis tubing with a LMW cut-off. We Dialyse at least 4x8h against excess PBS.
According to ThermoFisher, Expi293 medium is not compatible with the Ni-NTA purification system, see FAQ of the product.
(https://www.thermofisher.com/order/catalog/product/A1435103#/A1435103).
There is a patent filed by Life Technologies scientists who claimed adding 5 mM Ni++ helped recovering the his tag proteins from similar medium.
(https://patents.google.com/patent/WO2013082518A1/en)
@Christiane: Although the discussion seems to be out of time, maybe our INDIGO Ni material could be of interest for you or other users: It is compatible to chelators, such as EDTA, reductants, such as DTT, and protease inhibitors, such as phenanthroline, and should work with Expi293.
The link is:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/
It is possible to get a sample free of charge for testing.
Best, Roland
Thank you Roland. That is good to know fore future optimization still!
I can confirm that I was able to isolate secreted His-tagged proteins directly from conditioned media using INDIGO Ni-Agarose, without any pre-processing. The media was Gibco DMEM (11965092) and 10% FBS.
I used 250 ul of the 50% slurry. I did not quantify % yield - my proteins are secreted at very low levels.
- Badges
- Science method