Contaminants in protein purification.....help!
Posted 02 May 2013 - 10:10 AM
I have expressed a plant protein of about 25 kDa in E.Coli and purified it using Ni-NTA affinity chromatography as my protein is his-tagged.
After purification I got other bands at 75 KDa, 50KDa and 10 KDa. I have a strong band at 25kDa.
When I sequenced the contaminants they matched with my protein and also with E.Coli database. The scores and coverage with the E.Coli proteins were higher than my protein.
I tried everything...using 500mM NaCl, using 1% tween-20 and triton X-100, 10% ethanol, 1.5M salt in wash, 15mM mercaptoethanol.....everything. I use 20mM imidazole in my binding buffer and then wash with 40mM and 100mM imidazole. And then elute with 250mM imidazole. Even after washing with 100mM I still have those contaminants present.
What can I possibly do more to get rid of the contaminants?
And can E.Coli proteins interact so strongly with a protein that even after all these and heating my samples in SDS buffer and DTT I can still see those bands in my SDS-PAGE at the mentioned position?
Any suggestions on these would be highly appreciated.
Posted 02 May 2013 - 10:41 AM
To determine if they are degradation products (ie the 10kd) you can do a western blot and subsequent anti-his antibody. This will show what has the his-tag.The 50KDa band could be a dimer. I frequently observe dimers with high expression levels. The best way to remove these contaminants is by size exclusion chromatography.
A while back I was having the same problem. I started freezing the pellets before cell lysis, and I took great care (and time) to resuspend the pellet and lyse the cells. This better cell lysis led to the contaminant bands disappearing. Once I lysed the cells and did high-speed centrifigation, I hardly observed a pellet (cell wall, etc). This is a good indication that the lysis was done properly.
Posted 03 May 2013 - 05:20 AM
Posted 14 May 2013 - 09:52 AM
For improved expression, try using different expression vectors (e.g. pET vs pBad). Also consider a solubility tag, like GST, SUMO or GFP. Our lab commonly uses a cleavable n-terminal SUMO tag fusion to express our proteins. I do find that this can help tremendously with expression efficiency. Another option to consider is to have your gene codon optimized for e. coli expression since it is from plant. For a 25kDa protein, this probably wouldn't cost more than a couple hundred bucks (companies like DNA 2.0 and GenScript can do this).