Hello everyone,
I'm currently doing my master thesis and I would like to produce some proteins in HEK293T. The gene encoding my protein was placed in a gateway plasmid by LR reaction to add an HisTag and a MycTag (pEZYmyc-His : plasmid #18701 from AddGene)
The proteins I'm working on are secreted proteins (they display a signal peptide) of around 27-30kDa.
I would like to gather enough of my proteins for a further experiment but before I want to see if the proteins are actually expressed in the culture media. I carried out SDS-page (acrylamide gel 12% and colored with Coomasie Blue) and WesternBlots.
- For SDS-Page: I collected the culture media, centrifuged it to get rid of the remaining cells and then I dialyzed it against PBS (50mM KPO4 and 150mM NaCl) to remove the amino acids and small components of the culture media. After that, I purified the dialyzed product with Ni-NTA column (from Qiagen) and collected all the fractions (equilibration of the column with PBS (50mM KPO4, 150mM NaCl and 5mM imidazol); wash with PBS (50mM KPO4, 150mM NaCl and 20mM imidazol); first elution with PBS (50mM KPO4, 150mM NaCl and 250mM imidazol); second elution with PBS (50mM KPO4, 150mM NaCl and 500mM imidazol).
- For the WB, I tried just to load the culture media or I precipitate the proteins from 1ml of culture media in acetone and then resuspended the pellet in sample buffer.
In both cases, I got some troubles with the migration and the revelation of my gels/membranes (probably because of the big amount of BSA present in the culture media of my cells).
Does anyone have an idea, advice, or a nice method to produce secreted proteins in HEK293T cells?
Thanks in advance for your answers.
Thibault Masai
PS. do not hesitate to ask me for more details if needed.
There are resins available fro removing albumin and IgG from culture medium. Another option is to adapt the cells to grow in serum-free medium.
https://www.thermofisher.com/order/catalog/product/89875#/89875
https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/serum-free-media.html
As you have mentioned that the size of target protein 30kDa which is smaller than BSA (66kDa) so you should see two separate bands on SDS correspond to each, I would desalt sample in 20-50 mM buffer and concentrate it then give it a try on SDS, I doubt that the higher salt concentration (150 mM) used may cause this.
Hope this helps
Dear Thibault
the simplest solution is probably try to use the Expi293 expression system, which is derived from HEK293. It is based on media with out BSA and it allow high cell density in suspension and therefore high protein productivity.
Unfortunately this system is quite expensive, but if the import of this system in your laboratory is compatible with your budget, it is a great system.
You can find some more information about it at the following link
https://www.blogger.com/video.g?token=AD6v5dyTuEwDXD2ztCnLIFHkG06cCp1jaRuUHOGFtHdiTFnj-yS_TyCyPqzvi6RogJaSP_sXqbJnMDIYplkXODpfOc21BETPPh74J7FJuwMPxHW4X79mURcAl7-V0ApA8BQrGUAgcSc
on my BLog (ProteoCool)
Otherwise as Adam suggested, you can try to shift to an serum free medium, but you have to adapt your cells to growth on it, by gradually replacing the current media with
https://www.thermofisher.com/order/catalog/product/12338018?SID=srch-srp-12338018#/12338018?SID=srch-srp-12338018
good luck
Manuele
Hello again,
After more western blots, it turns out that my protein is localized in the lysate. Because I added a 6xHisTag to my protein, I used an antibody against that tag and besides my protein, there is a bunch of other proteins, with a higher MW, that is also detected.
As I plan to use a Ni column to purify my protein do you have any advice to get rid of those "contaminant" His-labelled proteins?
Thanks in advance,
Thibault
Unless your protein is expressed at a very high level, you will probably not be able to purify it with only a Ni column. Consider adding a second chromatography step, such as ion exchange or gel filtration.
Dear Thibault
i think that you have to understand the reason why the protein is not secreted but it localize in the lisate.
it is that its signal peptide is not work well in HEK293 (are your protein human protein? or derived from a different organism?) and you have to replace it with a standard one?
or the protein is not secreted because for some unknown reason the protein is not correctly folded and you have to re-desing the domain'? for example are the tags located at the N-term (close to signal peptide) or the C:terrm?
ciao
MAnuele
- Badges
- Science topic