Hello,
I'm trying to purify a 47 kD fusion protein by loading the supernatant directly on the column but I'm having issues with multiple species in my elutions.
The lanes are: ladder, supernatant, Wash1, W2, W3, elution 1, E2, E3, E4, E5, column.
The buffers for E1 and E2 are 10 mM reduced glutathione (RG), 50 mM tris, pH 8.
Elution 3 is an overnight on column incubation of 500 mM NaCl, 50 mM RG, 50 mM tris, pH 8.
The elution buffer for E4 and E5 are the same as E3 but are only 10' incubations.
The very last lane is a little bit of the column resuspended in PBS. This method gets all the protein off the column and into my elutions.
My problem is that I don't know how to get rid of the extra bands in E1 and E2 and get just my protein (darker bands more centered on gel).
So far I've tried: pre washing before elution with 50 mM tris pH 8, eluting with decreasing amounts of tris and RG at pH 8 and pH 9, eluting with different concentrations of NaCl (this is current elution method that isn't clean), and prewashing with 500 mM NaCl and 50 mM tris at pH8 but nothing has worked.
My mentor believes that these are chaperones which can be potentially removed with 2 mM ATP (this is from the glutathione spheres 4b booklet from GE healthcare) but I'm open to any ideas to try. Thanks a lot guys!
Hi there, you can just cut the band and extract the protein.
good luck
adelina
Hi there,
I guess there is a cleavage site between GST and your protein therefore instead of eluting GST fusion with GSH you may try on column digest with the specific protease. The main advantage is that GST would remain asociated to the matrix whereas efficient cleavage would allow elution of your protein.
Hi Benjamin,
About contaminant proteins :
1) you have one very fat band at around 25 kDa - I guess it could be free GST. At least I have faced with this problem few times when I expressed human proteins in E.coli, probably bacteria have a problem with transcription/translation after GST and you have both full-lenght (GST+POI) and just GST products. Since GST have tendency to dimerization, it is not easy to remove this contaminant (gel-filtration for example did not work well in this case). For purifing your GST-tagged protein you can use Heparin sepharose (at pH 7.4) or cation exchange resin like SP-sepharose (utilize low pI of GST).
2) Upper band probably chaperones, for washing out this contaminant you can wash your protein after binding to the resin with ATP/Mg2+ buffer, I used 25mM HEPES-NaOH pH 7.4 (RT), 100 mM KCl, 1 mM ATP, 1 mM MgCl2, 5 mM b-mercaptoethanol, 5% glycerol - let pass one CV, then add 1 CV and incubate 10 min, then wash with the next buffer.
In general no bad also to include one washing step with detergent, for example 0,05-0,1% TritonX100 (or NP40).
Hope it will help,
Gud luck with your protein purification!
Thank you all for your suggestions!
Adelina- this sounded like a good idea but I need at least a couple mL's of this protein so unfortunately this method won't work for me.
Dominique- thanks for your idea. I've already tried an on column digest however the protein precipitates out after cleavage.
Rafil- currently I'm using a glutathione spharose column to purify my POI. I did try washing with ATP and Mg.
Wash B: 2 mM ATP, 10 mM MgCl2, .1mg/mL denatured protein (idea from: D.V. Rial, E. A Ceccarelli, Removal of DnaK contamination during fusion protein purifications, Protein Expression and Purification, 25 (2002) 503-507)
EBA: 10 mM RG, 50 mM tris, pH 8
EBB: 500 mM NaCl, 50 mM NaCl, 50 mM tris, pH 8.
loading: For the protein loaded I pooled and dialyzed a couple E1 and E2 samples from the method used in original figure.
lanes: ladder, POI pooled and dialyzed, FT, W1 (3 mL PBS), W2 (3 mL PBS). W3 (3 mL PBS), W4 (1 mL Wash B), W5 (1 mL Wash B), W6 (1 mL Wash B). E1 (1 mL EBA), E2 (1 mL EBA), E3 (O/N 1.5 mL incubation EBB), E4 (10' 1 mL incubation EBB), E5 (10' 1 mL incubation EBB), E6 (10' 1 mL incubation EBB), column.
So it looks like the ATP/ Mg wash helped with removing DnaK slightly, but still not much cleaner than just taking the last three elutions. The bottom bands persist and I'd really like to remove those if you guys have any additional suggestions. I'm growing a new culture with 10 mM MgCl2 because I read from the Ceccarelli guy that it could help remove chaperonins. One of those bands down there has to be leaky GST expression I'm guessing.
Thanks everyone!
Benjamin,
For removing the free GST tag (lower band) from your protein eluates you can use cation exchange resin, like sp-sepharose (GE healthcare) or similar resins from another vendors (BioRad's UNOsphere S etc). GST protein has theoretical pI 5.90, if you will load sample on column in buffer with pH above this pI (more than 7 for being sure) GST tag will be negatively charged and will be in flow-through. Another resin that you can use is heparin-sepharose - it also kind of cation exchanger (for DNA binding protein also work as an affine resin).
But before check the pI of the your POI, if it positively charged at given pH, your protein will bind to the resin and you can elute it with high salt. Here is better to use step elution instead of gradient - you will have your protein more concentrated.
Given that the contaminating proteins are distinct in molecular weight (at least when denatured) from your fusion protein, a size exclusion column would also help as a second purification step.
The appearance of free GST is a problem that in my view can best be tackled by making alternative constructs that vary the distance between the protein and the tag. Too long linkers will attract proteases, too short linkers may interfere with folding of the individual parts. No safe predictions can be made so you need a trial and error approach.
Best wishes
Jürgen
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