May be this one helps....
"
The current study presents the design, engineering, and characterization of two
E. coli BL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the
endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C
terminus to a chitin binding domain (CBD) and the protein GlmS, with six
surface histidines replaced by alanines. "
http://www.ncbi.nlm.nih.gov/pubmed/21602383
Regards,Raj
Date: Wed, 18 Jan 2012 18:26:39 +0530
From: [email protected]
Subject: [ccp4bb] His Purification
To: [email protected]
Hello Every one,
I am trying to purify a human protein in a bacterial
expression system of around 82 kDa (with a 5 kDa His tag, so fusion protein is
87 kDa) which was cloned in pRSET-A vector. The Problem is I am not able to get
rid of the infamous contamination proteins of arnA gene (72 kDa) and glmS gene
(67 kDa).
I am using TEV protease to cleave my protein (82 kDa) from the tag (5 kDa).
This TEV protease has N- terminal His tag. So I first elute my fusion protein
with higher imidazole concentration and then do TEV cleavage by adding TEV
protease,, but sadly It co-elutes other contamination proteins such as 72 kDa
and 67 kDa, as mentioned above..
Now, I wanted to know,,, can I do "On beads cleavage" by directly adding TEV
enzyme when the fusion protein is still bound to Ni-NTA beads??
But I am worreid TEV protease which has N- terminal His tag also try to bind
on Ni-NTA beads..
Please help me..
Thanks!!
--
B4U
