Subject: Re: His tag does not bind.
From: hong yu honghyu {- at -} HOTMAIL {- dot -} COM
Date: 2007-10-11
150Kd is a fairly large protein for a MBP (55Kd) fusion. Six His-tag may not strong enough for it to bind to the Ni-column. If protein overexpression and solubility is not an issue, I would avoid using MBP fusion. Depend on how your protein fold, sometimes N-terminal His-tag works, sometimes C-terminal. If your N-terminal protein hide inside of the tertiary structure, try to clone it into a C-terminal His-tag vector. pET vectors offer easy switch. Good luck. Helen R&D/ABI
From: Kendall Nettles <knettles@SCRIPPS.EDU>
Reply-To: Kendall Nettles <knettles@SCRIPPS.EDU>
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] His tag does not bind.
Date: Thu, 11 Oct 2007 11:34:46 -0400
We have found that our His-MBP fusion doesn’t bind well after we cut off the protein of interest, and are trying to remove it. We have to use very low salt, cold temp, and slow loading rates. You might also try batch instead of column loading. We have also had good luck adding 1-2M urea to uncut His-MBP-protein fusions that show poor binding to the Qiagen Ni-NTA.
Kendall
On 10/10/07 9:12 PM, "changrui lu" <changrui@GMAIL.COM> wrote:
Dear all,
I am trying to express a 150 kd protein in E coli. I have it in two constructs, one with pmal-his and other with only his tag at N terminus. The full length protein can be detected both by sds and western using anti-his (190kd and 150kd respectively) but strangely neither binds to his-column very well. The majority of the full length comes through the column either at loading step or low salt wash step. The major species that gets trapped and eluted is the mbp-his truncation (~40kd). Some, though very little, full length protein did make it out the his column. The pmal-his construct does not bind amylose resin any better with majority flows right through. All purification are carried out under standard conditions as mentioned in the manuals. The protein is soluble and does not precipitate in
the columns. I appreciate and ideas or explanations.
Thanks in advance.
Ray
Cornell Univerisity
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