Hello all, our lab is trying to make a recombinant protein in E Coli using Ni-NTA chromatography. We have used our protocol many times successfully in the past but recently when we run our purified protein on SDS-PAGE, we are getting a single large band ~200kDa that seems to be a multimeric form of our protein. We are heating the samples 70C for 15 mins with detergents prior to loading our sample on the gel and the multimeric form still persists under these harsh conditions. We have replaced all of the reagents that we use, and have tried using lower amounts of starter culture and still we end up with the multimeric form.
Thank you
I would suggest to add 0,2M L-arginine in your loading and elution buffers. This concentration will prevent protein aggregation without interfering with the affinity chromatography step. Concentrations of L-arginine higher than 0,2M could strip the nickel from the colum, reduicing the final yelds of purification.
That might result from competition of SDS with some other detergents (such as Nonidet P-40) in your sample. Especially, this can be revealed with commercial gels which often don't have SDS in the gel itself. You might want to increase the concentration of SDS before boiling (3%-4%). I had such problem in the past.
Hi there,
I would suggest you to express the protein at low temperature with (Chaperone Induction) condition to provide proper condition for protein folding; before inducing the culture (E.coli) subject them to 42 degree celusis temperature condition followed by 18 degree induction with IPTG for 16 hours.
I hope this was useful.
Rajkumar
If your problem with aggregation is only occurring when you are preparing your samples for SDS-PAGE analysis, I would suggest not heating your samples prior to loading. Try incubating at room temp (time frame 30 min to several hours depending on protein)
If you suspect that aggregation of your protein of interest occurs after purification, you may consider adding BSA in your stock (if it is compatible with further uses of your protein)
Hi there
I would defintely denature much more aggressively than 70C for 15 min. Routinely I would used to use 5 min at 100c in the presence of b ME but 10 min is meant to help in these situations. In addition make sure you snap Cool having incubated for 10 min at 100c. This will ensure no renaturation plus concatemetisation following more gradual cooling
Hi Kelsey
Are you sure that 200 K band is really your protein? Did you perform western? Does your protein contains unpaired cysteines?
In any case heating at 100C in 2% SDS and 5% 2ME would disrupt any protein aggregates. By the way, 70C is unsufficient to complete disruption of disulfide bonds.
Best regards,
Nikolay.
To prevent aggregation of the purified protein of interest, try adding some sugars like sorbitol or sucrose; Detergent like polysorbate 20 or 80; amino acid like arginine etc to the purified protein solution after final purification of the same. This would definitely solve your issue.
Hi
You can try denature proteins with 6 M urea before to add sample buffer to SDS-PAGE.
The sample with 6M urea and sample buffer (0.06 M Tris-HCl , pH 6.8, 2% SDS, 10% glycerol, 0.025% bromophenol blue, 5% 2-mercaptoethanol) (ratio 1:1) heat up at 95°C for 10 min and immediately put the sample on ice. Centrifuge the sample and run SDS-PAGE.
I had similar problem when I expressed recombinant protein in P. pastoris. If your protein has not enzymatic activity, you can use denaturation by urea, but if have enzymatic activity you need refolding the protein by dialysis. Urea method is only to characterize by SDS-PAGE.
The concentration of urea must not interfere with SDS-PAGE running, the salt concentration does not affect, this quantities are used for 2D-PAGE.
Thanks so much for the responses.
We ran a gel of our lysed cells and even then we are getting aggregation right off the bat. Western blot confirms that it is in fact our protein of interest. We had always used the same SDS-PAGE conditions in the past and were able to detect the monomeric form so we do believe this aggregation is happening before the purification process.
I would suggest to add 0,2M L-arginine in your loading and elution buffers. This concentration will prevent protein aggregation without interfering with the affinity chromatography step. Concentrations of L-arginine higher than 0,2M could strip the nickel from the colum, reduicing the final yelds of purification.
Hi Wong,
Does your protein of interest has tendency to form Multimer in native condition (In Nature) ? Denaturing PAGE will help you to resolve this issue. Whether the purification tag is attached to N-terminus or C-terminus? If it is C-terminus I suggest you to check for intact stop codon after the purification tag. Sometime the whole issue can resolved by shifting C-terminal tagging to N-terminal (if you protein activity is not compromised).
Thanks again for all of the wonderful insight.
The His tag is at the N terminal of our protein and in fact our protein does have tendency to form multimers under native conditions. We have isolated this protein as a monomer in the past many times, however we have gotten multimer the last 5-10 times or so. We have tried the various denaturing steps prior to SDS-PAGE and still we are getting the multimer, which seems to be irreversible.
Although a bit late, I would like to add my two cents to this discussion.
Make sure that your loading buffer for SDS-PAGE is fresh and reducing enough. Old beta-ME or DTT can lead to less reducing sample buffer. We are using following buffer (2x)
125 mM Tris-H3PO4 (pH 7.5 at 25°C)
2 mM EDTA
4 % SDS
200 mM DTT
2 mM NaN3
0,01 % Bromophenol Blue
50 % Glycerol
Note that the suggestion of boiling the samples in 100°C for some time would always be better than your way of 70° for 15min can not be generalized. It is not true that boiling will always lead to a reduction of aggregates in the gel. In fact, with some proteins that have already a high tendency to form aggregates and especially membrane proteins with highly hydrophobic domains (not necessarily due to disulfide bonds), you will even get more aggregates at 100°C! In such a case heating the samples at lower temp. of 50 -70°C might even be the better option.
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