Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
- - - - -

Contaminants in protein purification.....help!

  • Please log in to reply
3 replies to this topic

#1 Bioscientist



  • Active Members
  • Pip
  • 26 posts

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.






  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 159 posts

Posted 02 May 2013 - 10:41 AM

A couple of things...

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.

#3 Missle



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 166 posts

Posted 03 May 2013 - 05:20 AM

It is difficult to avoid having at least some contaminating E.coli host cell proteins when purifying the soluble lysate using Ni-NTA chromatography. I've found if you're doing all the logical things (altering the imidazole and NaCl concentration of binding/washing, etc...), then the purity is largely due to your expression level - the more of your tagged protein present in the sample the higher you're purity will be. If you need a higher purity, you should consider a second purification step. Either size exclusion and ion exchange chromatography are good choices. They require more work but they'll get you to the purity you need much faster than trying everything you've been trying.....

Good luck!

#4 labtastic



  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 205 posts

Posted 14 May 2013 - 09:52 AM

Missle is spot on...if you've tried all those basic tricks, the impurities are largely due to poor expression. The other option, as HOYAJM eluded to, is that they may be degradation products, in which case throw in some protease inhibitors during lysis. I also like to use a C-terminal his-tag on my protein, that way I know I am never purifying translationally truncated peptides as you would if you used an n-terminal his-tag.

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).

Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.