E coli expression of eucaryotic protein

[QIAGEN]

In article <kallisti-1707952017580001 at lomasneylab.feinberg.nwu.edu>,
kallisti at merle.acns.nwu.edu (Patrick Grealish) wrote:

> HEllo, I am current starting another experiment where i'll need to produce
> eucarotic protein in E. coli. i have used in the past the Ni-agarose
> column method (ie quiagen) but i've found that even with careful column
> washiung i was still getting some cotamination (kind of alot actually)
> some i was hoping to utilize a different method of tagging (not the 6Xhis
> tag). If anyone can help out i would greatly appreciate the assistance.
> TIA
> 
> -- 
> hail eris all hail discordia kallisti
> Patrick Grealish
> kallisti at merle.acns.nwu.edu


Hi Patrick,
this is in reference to your e-mail regarding purification problems with
Ni-
NTA Agarose.
I am the product manager for the QIAexpress product line and just wanted to

mention a couple of things that other customers have used successfully to
get 
their 6xHis-tagged proteins over 95% pure in just one pass over the Ni-
NTA column. Whether you have incorporated these steps in your purification 
already or not, but would like to give it another try, please do not
hesitate 
to call me or technical service at 800-426-8157.

Suggestions: 
1. binding in batch, instead of on the column, for better binding kinetics
2. adding 10 mM §-ME in the lysis, binding and wash steps to avoid
disulfide 
bridges from forming (i.e. between your protein and E. coli host proteins)
3. adding up to 20 mM imidazole in the binding step to initially avoid any 
non-specific binding of host proteins to the resin
4. further the resin is stable  in 6 M GuHCl, 8 M urea, 2% Triton or Tween,

1% CHAPS, 2 M NaCl, 50% glycerol and 30% ethanol, which might be helpful.

Regards,
Brigitte Steude Masone

-- 
QIAGEN       800-362-7737
Temporary Email Address          qiagen at kaiwan.com