Have you tried dialyzing the "purified" protein after elution into a no
imidazole buffer while doing TEV cleavage, and then reloading on to a His
column to remove the tag and TEV? I've found in my experience that this often
recaptures a lot of the background proteins.
If you're getting a 70kDa
my comment on expression of protein being low
and nonspecific binding.
Amount of protein of expressed protein less = less resin = less
nonspecific binding?
(one have to do experiments to
find right amount of resin to get least non-specific binding
still pull out most of your protein of interest is
Your mileage will vary: prset is an almost always quasi constituitive
vector due to leakage, with additional induction upon expression of T7 pol.
This is not always bad since some proteins do not fold well upon avalanche
inducton but do in fact fold when expressed at a trickle (even good old lac
pr
avoid pRSET would be my recommendation - my impression from a couple of
examples is pRSET gives badly folded protein.
Can you easily subclone it in a better expression vector?
(personally, even if it were not so easy, I would reclone it in another
expression vector).
Mark J van Raaij
Laboratorio
ect: [ccp4bb] His Purification
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
Another option is to try NEB NiCo21(DE3) cells.
http://www.neb.com/nebecomm/products/productC2529.asp
I have no relation to NEB beyond being a customer.
They've mutated GlmS to eliminate binding to IMAC resins and have
added chitin affinity tags to SlyD, ArnA and Can to allow simple post-
I
nding 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: bhanu.hydpri...@gmail.com
Subject: [ccp4bb] His Purification
To: CCP4BB@JISCMAIL.AC.UK
Hello
On Wed, 2012-01-18 at 18:26 +0530, PULSARSTRIAN wrote:
> 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).
This is only a problem if you plan to have imac purification as your
only step. If the goal is crystallization,
pays off now **
- Original Message -
From: "PULSARSTRIAN"
To: CCP4BB@JISCMAIL.AC.UK
Sent: Wednesday, January 18, 2012 7:56:39 AM
Subject: [ccp4bb] His Purification
Hello Every one,
I am trying to purify a human protein in a bacterial expression system of
around 82 kDa (wi
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
10 matches
Mail list logo
