Hello all:
I'm trying to isolate a protein I am expressing in CHO cells. The protein is secreted into media and the two isoforms are either 20 kda or 30 kda. However, there are serum proteins I'd like to get rid off, north of 70 kDa. Are there any size exclusion techniques (i.e. chromatography) that I could use to get rid of these larger proteins? Thank you.
Humble Lab Tech!
Dear Anant
if your protein are monomeric and therefore run as 20-30 kda in size exclusion you can certanilly separate it from a 70kda protein using supedex 75 coloumn ( 16/60 or 26/60 depending of the amount of sample that you would like to load.
however you can solve the problem also:
- adapting your cells in a serum free media
- adding a histidine tag to your protein and perform an imac purification.
best regards
Manuele
Manuele martinelli, I recommend this one,
And if you want to isolate a protein from a whole lysate ,you can go with the size exclusion i added the pic which as a molecular cutoff range. If you have histidine tag you can do affinity chromatography using NI-NTA
I would perform a purification step like His-Tag purification as Manuele Martinelli suggested to increase purity and concentration. However lots of serum proteins also bind to Ni-NTA matrix, so working serum free is a good idea to further improve purity, but can impair protein yield.
There are spin columns for protein concentration (Amicon for example) with a cutoff at a certain molecular weight. You could use a 50 kDa cutoff column first, collect your flow-through and concentrate it further via a 10 kDa cutoff column. Your concentrate should only contain proteins in the range of 10-50 kDa then, eliminating most of your impurities.
Good luck,
Jan
Regarding the serum free medium, you can try the Free style CHO from thermo
https://www.thermofisher.com/order/catalog/product/12651014?SID=srch-srp-12651014
https://assets.thermofisher.com/TFS-Assets/LSG/manuals/5002_FreestyleCHO_PI.pdf
Generally Thermo mediums are expensive but works quite well.
They sold also a powerfull version of CHO that are named Expi-CHO that guarantee very high expression levels, but its are very very expensives!
good luck
Manuele
Thank you all for your assistance. I sincerely appreciate it. I do have a version of the protein I'm trying to overexpress that has a n terminal 9xhis tag. It also has a signal peptide that should secrete it into the medium. The problems are as follows: 1) As someone rightly pointed out, serum proteins that bind nickel resin. We are using adherent cho k1 cells and 2 percent fbs. Unfortunately, that's enough to foul things. 2) The signal peptide seems suboptimal. Either the protein gets jammed in the cytoplasm or comes out improperly folded. 3) The his tag seems hidden. So, there are multiple issues to solve. Also, does anyone think there's benefit to adding edta free protease inhibitors to media that the protein is being secreted into? Sorry for the multiple questions but thank you all for your expertise.
May I ask what kind of signal peptide you use? I have had good secretion results with the IgK Leader in HEK cells, however I have no experience with it in CHO cells.
Regarding the His Tag: Maybe you can put a small flexible linker like GGGS between the tag and your protein, or try tagging your protein at the C-terminus instead. It could be that your His-Tag at the N-terminus interferes with the secretion peptide.
Cheers,
Jan
I agree with Jan Philipp Dobert . But, since modifying the construct would take a lot of effort, I would suggest trying ion exchange to concentrate your target protein prior to size exclusion. Hopefully, it can also partially purify your target protein.
Hi Anant,
I agree with Yanuar Sebastian Tandra. If your protein is a monomer, gel filtration (superdex75) and ion exchange are enough to isolate the target protein.
All the best!
Gel filtration (size exclusion chromatography, SEC) is a non-concentrating technique, the protein peak always has a larger volume than the sample peak you added to the column. The larger the sample volume, the more the peaks overlap. Therefore, SEC is used as the last step in a purification, with a highly concentrated sample.
The first step should be a concentrating technique like affinity chromatography, IEC or HIC. If your affinity tag doesn't work for whatever reason, you can still try any of the other methods.
Of course, you need to acertain first that the protein is actually secreted, a simple SDS-PAGE (perhaps followed by WB with anti-His6 antibodies) can do that.
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