I have purified a protein, however I am getting double band on SDS-PAGE. Even after FPLC the double band doesn't resolve and become more prominent. I have checked through Mass spec that both the band are of my protein of interest however one of the band indicates cleaved N term of protein. The buffer condition is normal Tris- NaCl; pH 7.5 and the size of protein is 9.56kD. Please suggest me how can I prevent this as I want to crystallise the protein.
The N-terminal amino acids, in particular Met-1, are usually removed by cellular processing. Perhaps this process is incomplete. Since you have access to MS/MS, this is easy to verify.
Your protein might have a auto-proteolitic activity. Is the amount of cleaved protein the same as the non-cleaved? Try to crystallize it anyway, you might have some good results.
Engelbert Buxbaum my protein domain is starting from glycine after the His-Tag and TEV site.
José Malanho Silva The domain I am working with is SAM domain so auto-proteolytic activity would be quite rare. The cleaved portion amount for around 4 peptides and non cleaved 28 as per mass spec data.
It could be the Glycosylated and non-glycosylated protein. As I always get this "double trouble" band for my protein. Please note that we are working with insects cell expression.
I agree with Saima Rehman. If your protein is from a eukaryotic expression system: We have the same case because of the heterogeneity in glycosylation. After treating the sample with endoglycosidase (eg. Endo H) we could observe a single band instead of two bands. If not, for a bacterial expression system it could then be because of degradation. May I know your expression system?
Udaya kumar Tiruttani subhramanyam Expression system is bacterial and I have checked through MS that the cleaved baned is not glycosylated but N terminal portion.
Ok. Hoping that you are using protease-inhibitors, I would suggest to use them throughout the purification. I also would suggest to use cocktail inhibitors instead of a single protease inhibitor. Did you observe these two bands after TEV cleavage or they are there even before? if not, I recommend José Malanho Silva's crystallization-suggestion because I haven't seen any 3D structure showing his-tag (Maybe heterogeneity in His-tag may not effect your crystal packing). Good luck.
Udaya kumar Tiruttani subhramanyam I am using cocktail inhibitors as well to this I have tried to purify proteins with and without TEV cleavage. However, I have not find any change with respect to the doublet.
Hi Shubhashish,
Have you tried the ion exchange? If the protein is evenly charged, you might try to crystallize fractions that corresponds to each peak respectively. Good luck.
Hi Shubhashish Chakraborty ,
From your information, I suspect that both proteins were structurally similar or even interact with each other, thus co--purify during the process. Ion exchange would be good if the cleaved protein lost two or more ionic residue (from my experience, smaller protein will be more sensitive to charge difference during chromatography). If both protein interact with each other, you might want to try adding mild chaotropic agent to your eluent (ethanol, 0.5-1 M urea, depends on your protein stability) to disrupt the interaction between cleaved and the intact protein.
Good luck!
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